Increased Responsiveness of Stored Platelets after Short-Term Refrigeration.

Introduction: Refrigeration of platelets is considered to provide advantages in therapy of acute hemorrhage due to increased platelet responsiveness. The alleviation of inhibitory signaling caused by cold temperature (CT) has been identified as an important mechanism contributing to enhanced platelet reactivity, detectable in freshly prepared platelets within 1 h of cold storage. The aim of this study was to confirm the effects of short-term refrigeration in platelets from apheresis-derived platelet concentrates (APC). Methods: APC were stored under standardized conditions for 1 day or for 2 days at room temperature and then refrigerated for 1 h, followed by sampling of platelets for analysis. Platelet reactivity was measured by aggregation studies using threshold concentrations of different agonists and by detection of fibrinogen binding using flow cytometry. The exploration of inhibitory signaling comprised the detection of VASP phosphorylation using flow cytometry or Western blot and the measurement of cyclic nucleotide levels. Results: Aggregation responses induced with ADP, collagen, or thrombin receptor-activating peptide-6 (TRAP-6) were increased in APC after cold storage for 1 h, associated with elevated TRAP-6-induced fibrinogen binding. VASP phosphorylation levels were decreased after cold exposition, detectable in 1-day- and 2-day-stored APC with flow cytometry, and in 2-day-stored APC with Western blot technique. Induced cGMP levels were lower after storage at CT in APC on day 1 and on day 2, whereas cAMP levels were reduced on 2-day-stored APC. Conclusion: Short-term refrigeration for 1 h is sufficient to induce an attenuation of inhibitory signaling, accompanied with increased aggregation responses in APC stored for up to 2 days. The "on demand" refrigeration of PC may be a reasonable approach for the preparation of platelets with enhanced responsiveness to treat patients with hemorrhage more effectively, which should be further addressed in consecutive studies.

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence.

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

Bare Stent Fracture After TEVAR With the Modified Restrictive Bare Stent (RBS) Technique in Type B Aortic Dissections.

PURPOSE: The aim was to assess the mid-term aortic remodeling and bare-metal stent (BMS) integrity of the restricted bare stent (RBS) technique reconstruction in aortic dissections. MATERIALS AND METHODS: This retrospective cohort study included prospectively collected patients treated with the modified RBS technique between 2017 and 2020. The preoperative, postoperative, and last follow-up computed tomographic (CT) scans were analyzed in the centerline at the mid-descending, celiac trunk (CeT), and the mid-abdominal levels for false lumen (FL) patency, aortic diameter, and true lumen (TL) diameter changes. Bare-metal stent integrity was assessed in the 3-dimensional multiplanar reformats. RESULTS: The median follow-up of the cohort (n=17) was 26 (11, 45) months. The procedure was mainly performed with the Relay NBS endograft (15/17; 88%) + E-XL BMS (17/17; 100%). Postoperative mortality, paraplegia, stroke, renovisceral vessel loss, and type I and III endoleaks were not observed. BMS fractured in 6 patients (6/17; 36%), damaged the dissection flap in 4/17 (24%), and led to the reperfusion of the FL and re-interventions with TEVAR (4/17; 24%). Two patients without FL reperfusion showed stable CT follow-ups 13 and 17 months after the fracture diagnosis. The TL expansion was seen at all landmarks and peaked in the thoracic aorta (+10; 6, 15; p<0.001). The FL thrombosis after modified RBS was only relevant in the thoracic aorta (p<0.001) and at CeT (p=0.003). The aortic diameter was stable in the thoracic aorta and increased at distal landmarks (CeT [+5; 1, 10; p=0.001]; mid-abdominal [+3; 1, 5; p=0.004]). CONCLUSION: The modified RBS technique could not stop aortic growth below the diaphragm and prevent new membrane rupture due to the fractures of the BMS and consecutive flap damage with the reperfusion of the FL. CLINICAL IMPACT: The treatment of complicated type B aortic dissections with TEVAR has become a standard. Particularly, patients with true lumen collapse and malperfusion may benefit from a more aggressive treatment strategy including proximal TEVAR and distal bare-metal stent implantation to re-open the true lumen and to prevent distal stent-induced new entry. However, this study reports the challenges of this approach with a high rate of bare-metal stent fractures during the follow-up. The fractures that occurred at the site of vertical nitinol bridges led to the dissection membrane ruptures and the reperfusion of the false lumen with consecutive dilatation. A close follow-up is mandatory to detect this complication and to treat the patients with TEVAR extension.

The impact of 3D printed models on spatial orientation in echocardiography teaching.

PURPOSE: During our transthoracic echocardiography (TTE) courses, medical students showed difficulty in spatial orientation. We implemented the use of 3D printed cardiac models of standard TTE views PLAX, PSAX, and A4C and assessed their efficacy in TTE-teaching. METHODS: One hundred fifty-three participants were split into two groups. A pre-test-retest of anatomy, 2D -, and 3D orientation was conducted. The intervention group (n = 77) was taught using 3D models; the control group (n = 76) without. Both were comparable with respect to baseline parameters. Besides test-scores, a Likert scale recorded experiences, difficulties, and evaluation of teaching instruments. RESULTS: From the 153 students evaluated, 123 improved, 20 did worse, and ten achieved the same result after the course. The median overall pre-test score was 29 of 41 points, and the retest score was 35 (p < 0.001). However, the intervention group taught with the 3D models, scored significantly better overall (p = 0.016), and in 2D-thinking (p = 0.002) and visual thinking (p = 0.006) subtests. A backward multivariate linear regression model revealed that the 3D models are a strong individual predictor of an excellent visual thinking score. In addition, our study showed that students with difficulty in visual thinking benefited considerably from the 3D models. CONCLUSION: Students taught using the 3D models significantly improved when compared with conventional teaching. Students regarded the provided models as most helpful in their learning process. We advocate the implementation of 3D-printed heart models featuring the standard views for teaching echocardiography. These findings may be transferable to other evidence based medical and surgical teaching interventions.

T3P-Promoted Synthesis of a Series of 2-Aryl-3-phenyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-ones and Their Activity against the Kinetoplastid Parasite Trypanosoma brucei.

A series of fourteen 2-aryl-3-phenyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-ones was prepared at room temperature by T3P-mediated cyclization of N-phenyl-C-aryl imines with thionicotinic acid, two difficult substrates. The reactions were operationally simple, did not require specialized equipment or anhydrous solvents, could be performed as either two or three component reactions, and gave moderate-good yields as high as 63%. This provides ready access to N-phenyl compounds in this family, which have been generally difficult to prepare. As part of the study, the first crystal structure of neutral thionicotinic acid is also reported, and showed the molecule to be in the form of the thione tautomer. Additionally, the synthesized compounds were tested against T. brucei, the causative agent of Human African Sleeping Sickness. Screening at 50 µM concentration showed that five of the compounds strongly inhibited growth and killed parasites.

Sleep enhances numbers and function of monocytes and improves bacterial infection outcome in mice.

Sleep strongly impacts both humoral and cellular immunity; however, its acute effects on the innate immune defense against pathogens are unclear. Here, we elucidated in mice whether sleep affects the numbers and functions of innate immune cells and their defense against systemic bacterial infection. Sleep significantly increased numbers of classical monocytes in blood and spleen of mice that were allowed to sleep for six hours at the beginning of the normal resting phase compared to mice kept awake for the same time. The sleep-induced effect on classical monocytes was neither caused by alterations in corticosterone nor myelopoiesis, bone marrow egress or death of monocytes and did only partially involve Gαi-protein coupled receptors like chemokine receptor 2 (CCR2), but not the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) or lymphocyte function-associated antigen 1 (LFA-1). Notably, sleep suppressed the expression of the clock gene Arntl in splenic monocytes and the sleep-induced increase in circulating classical monocytes was abrogated in Arntl-deficient animals, indicating that sleep is a prerequisite for clock-gene driven rhythmic trafficking of classical monocytes. Sleep also enhanced the production of reactive oxygen species by monocytes and neutrophils. Moreover, sleep profoundly reduced bacterial load in blood and spleen of mice that were allowed to sleep before systemic bacterial infection and consequently increased survival upon infection. These data provide the first evidence that sleep enhances numbers and function of innate immune cells and therewith strengthens early defense against bacterial pathogens.

The assembly and evaluation of antisense oligonucleotides applied in exon skipping for titin-based mutations in dilated cardiomyopathy.

The leading cause of genetic dilated cardiomyopathy (DCM) is due to mutations in the TTN gene, impacting approximately 15-20% of familial and 18% of sporadic DCM cases. Currently, there is potential for a personalized RNA-based therapeutic approach in titin-based DCM, utilizing antisense oligonucleotide (AON) mediated exon-skipping, which attempts to reframe mutated titin transcripts, resulting in shortened, functional protein. However, the TTN gene is massive with 363 exons; each newly identified TTN exon mutation provides a challenge to address when considering the potential application of AON mediated exon skipping. In the initial phase of this strategy, the mutated TTN exon requires specific AON design and evaluation to assess the exon skipping effectiveness for subsequent experiments. Here, we present a detailed protocol to effectively assemble and evaluate AONs for efficient exon-skipping in targeted TTN exons. We chose a previously identified TTN 1-bp deletion mutation in exon 335 as an exemplary target exon, which causes a frameshift mutation leading to truncated A-band titin in DCM. We designed two specific AONs to mask the Ttn exon 335 and confirmed successfully mediated exon skipping without disrupting the Ttn reading frame. In addition, we evaluated and confirmed AON-treated HL-1 cells show maintained store-operated calcium entry, fractional shortening as well as preserved sarcomeric formation in comparison to control samples, indicating the treated cardiomyocytes retain adequate, essential cell function and structure, proving the treated cells can compensate for the loss of exon 335. These results indicate our method offers the first systematic protocol in designing and evaluating AONs specifically for mutated TTN target exons, expanding the framework of future advancements in the therapeutic potential of antisense-mediated exon skipping in titin-based DCM.

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor studies.

BACKGROUND: Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. METHODS: Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. RESULTS: Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. CONCLUSION: The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites' redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.

Conserved small mRNA with an unique, extended Shine-Dalgarno sequence.

Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3'-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.

Dysregulated IER3 Expression is Associated with Enhanced Apoptosis in Titin-Based Dilated Cardiomyopathy.

Apoptosis (type I programmed cell death) of cardiomyocytes is a major process that plays a role in the progression of heart failure. The early response gene IER3 regulates apoptosis in a wide variety of cells and organs. However, its role in heart failure is largely unknown. Here, we investigate the role of IER3 in an inducible heart failure mouse model. Heart failure was induced in a mouse model that imitates a human titin truncation mutation we found in a patient with dilated cardiomyopathy (DCM). Transferase dUTP nick end labeling (TUNEL) and ssDNA stainings showed induction of apoptosis in titin-deficient cardiomyocytes during heart failure development, while IER3 response was dysregulated. Chromatin immunoprecipitation and knock-down experiments revealed that IER3 proteins target the promotors of anti-apoptotic genes and act as an anti-apoptotic factor in cardiomyocytes. Its expression is blunted during heart failure development in a titin-deficient mouse model. Targeting the IER3 pathway to reduce cardiac apoptosis might be an effective therapeutic strategy to combat heart failure.

Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation.

BACKGROUND: Differential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters. RESULTS: A specialized machine learning procedure for TSS recognition allowed us to map 15,923 TSSs: 14,360 in free-living bacteria, 4329 in symbiosis with soybean and 2766 in both conditions. Further, we provide proteomic evidence for 4090 proteins, among them 107 proteins corresponding to new genes and 178 proteins with N-termini different from the existing annotation (72 and 109 of them with TSS support, respectively). Guided by proteomics evidence, previously identified TSSs and TSSs experimentally validated here, we assign a score threshold to flag 14 % of the mapped TSSs as a class of lower confidence. However, this class of lower confidence contains valid TSSs of low-abundant transcripts. Moreover, we developed a de novo algorithm to identify promoter motifs upstream of mapped TSSs, which is publicly available, and found motifs mainly used in symbiosis (similar to RpoN-dependent promoters) or under both conditions (similar to RpoD-dependent promoters). Mapped TSSs and putative promoters, proteomic evidence and updated gene annotation were combined into an annotation file. CONCLUSIONS: The genome-wide TSS and promoter maps along with the extended genome annotation of B. japonicum represent a valuable resource for future systems biology studies and for detailed analyses of individual non-coding transcripts and ORFs. Our data will also provide new insights into bacterial gene regulation during the agriculturally important symbiosis between rhizobia and legumes.

Mononuclear phagocytes contribute to intestinal invasion and dissemination of Yersinia enterocolitica.

Enteropathogenic Yersinia enterocolitica (Ye) enters the host via contaminated food. After colonisation of the small intestine Ye invades the Peyer's patches (PPs) via M cells and disseminates to the mesenteric lymph nodes (MLNs), spleen and liver. Whether Ye uses other invasion routes and which pathogenicity factors are required remains elusive. Oral infection of lymphotoxin-ß-receptor deficient mice lacking PPs and MLNs with Ye revealed similar bacterial load in the spleen 1h post infection as wild-type mice, demonstrating a PP-independent dissemination route for Ye. Immunohistological analysis of the small intestine revealed Ye in close contact with mononuclear phagocytes (MPs), specifically CX3CR1(+) monocyte-derived cells (MCs) as well as CD103(+) dendritic cells (DCs). This finding was confirmed by flow cytometry and imaging flow cytometry analysis of lamina propria (LP) leukocytes showing CD103(+) DCs and MCs with intracellular Ye. Uptake of Ye by LP CD103(+) DCs and MCs was dependent on the pathogenicity factor invasin, whereas the adhesin YadA was dispensable as demonstrated by Ye deletion mutants. Furthermore, Ye were found exclusively associated with CD103(+) DCs in the MLNs from wild-type mice, but not from CCR7(-/-) mice, demonstrating a CCR7 dependent transport of Ye by CD103(+) DCs from LP to the MLNs. In contrast, dissemination of Ye to the spleen was dependent on MCs as significantly less Ye could be recovered from the spleen of CX3CR1(GFP/GFP) mice compared to wild-type mice. Altogether, MCs and CD103(+) DCs contribute to immediate invasion and dissemination of Ye. This together with data from other bacteria suggests MPs as general pathogenic entry site in the intestine.

Intramural hematoma in the proximal sealing zone of the thoracic endovascular aneurysm repair: frequency and safety in acute and subacute type B dissections.

Introduction: To assess the outcomes after thoracic endovascular aneurysm repair (TEVAR) in the presence of intramural hematoma (IMH) in the proximal sealing zone. Material and methods: Patient data were retrospectively extracted from the hospital records of patients treated with TEVAR for acute and chronic aortic dissection type B in one single center. The initial, preoperative, first postoperative, and last follow-up CT scans were evaluated in the aortic 3D multiplanar reformats and the centerline regarding IMH presence in the proximal sealing zone, anatomical preconditions, and the morphological TEVAR complications including migration and bird-beak. Groups with (IMH) and without IMH (no-IMH) were compared. Results: Overall, 84 patients (IMH:42; no-IMH:42) were treated at the age of 63(55; 72) years, of whom 23/84 (27%), 34/84 (40%), and 27/84 (32%) were in the hyperacute, acute and subacute dissection phases, respectively. The bovine arch was found in 10/84(12%) and the type III arch was most common (43/84;51%). IMH maximum extent was found in zones 0, 1, 2, and 3 in 14/84 (17%), 17/84 (20%), 18/84 (21%), and 6/84 (7%), respectively. Sealing was achieved in zone II in 71/84 (85%) and LSA was revascularized in 66/84 (79%) of the overall cohort. Early mortality and paraplegia were 2/84 (2%) each; stroke rate was 3/84 (4%). During the 22 months median follow-up (22;4;43) no RTAD was observed. Migration ≥10 mm (IMH: 11/82; no-IMH: 10/82; P = 1.0) and bird-beaks (IMH: 10/82; no-IMH: 12/82; P = 0.8036) were comparable in both groups and accompanied by a low aorta related mortality (1/82) in both groups. Conclusion: The presence of the IMH in the proximal TEVAR sealing zone is frequent and may not be relevant for the occurrence of the RTAD, stent-graft migration, or bird-beak formation.

Mid-Term Outcomes of a Pre-Cannulated Iliac Branched Device in the Treatment of Abdominal Aortoiliac Aneurysms: A Retrospective Analysis from a Single Center.

The aim was to assess the mid-term results of the E-iliac branched device. Baseline and follow-up data of this monocentric retrospective cohort study including all consecutive patients with aortoiliac aneurysms treated with iliac branched devices between 2016 and 2023 were extracted from the hospital records. Preoperative and follow-up CT scans were analyzed regarding endoleaks, migration, aneurysm sac remodeling, and device patency. Overall, 50 devices were implanted in 38 patients with a median age of 69 (IQR 62-78) years, and 1.6 bridging stent grafts per vessel were implanted through transfemoral (22/50; 44%) or upper extremity access (28/50; 56%). Primary technical success and assisted technical success were 97% (37/38) and 100% (38/38), respectively. No migration, no type I or III endoleaks, no stroke, colonic ischemia, aneurysm rupture, or conversion during the early and mid-term follow-ups (11 months, IQR 5-26) were observed. Aneurysm sac enlargement or shrinkage was observed in 0% (0/38) and 16% (6/38) patients, respectively. E-iliac-related re-interventions were seen only during the early follow-up: two thrombectomies with bare-metal stent relining after thrombosis of the iliac limb. Bridging stent graft and E-iliac patency during the mid-term follow-up were 100%. E-iliac showed encouraging mid-term results in the treatment of aortoiliac aneurysms with high technical success and a low re-intervention rate.

Left Atrioventricular Transvalvular Pressure Gradients Derived from Intraoperative and Postoperative Echocardiograms following Atrioventricular Septal Defect Repair.

BACKGROUND: Left atrioventricular valve (LAVV) stenosis following an atrioventricular septal defect (AVSD) repair is a rare but potentially life-threatening complication. While echocardiographic quantification of diastolic transvalvular pressure gradients is paramount in the evaluation of a newly corrected valve function, it is hypothesized that these measured gradients are overestimated immediately following a cardiopulmonary bypass (CPB) due to the altered hemodynamics when compared to postoperative valve assessments using awake transthoracic echocardiography (TTE) upon recovery after surgery. METHODS: Out of the 72 patients screened for inclusion at a tertiary center, 39 patients undergoing an AVSD repair with both intraoperative transesophageal echocardiograms (TEE, performed immediately after a CPB) and an awake TTE (performed prior to hospital discharge) were retrospectively selected. The mean (MPGs) and peak pressure gradients (PPGs) were quantified using a Doppler echocardiography and other measures of interest were recorded (e.g., a non-invasive surrogate of the cardiac output and index (CI), left ventricular ejection fraction, blood pressures and airway pressures). The variables were analyzed using the paired Student's t-tests and Spearman's correlation coefficients. RESULTS: The MPGs were significantly higher in the intraoperative measurements when compared to the awake TTE (3.0 ± 1.2 vs. 2.3 ± 1.1 mmHg; p < 0.01); however, the PPGs did not significantly differ (6.6 ± 2.7 vs. 5.7 ± 2.8 mmHg; p = 0.06). Although the assessed intraoperative heart rates (HRs) were also higher (132 ± 17 vs. 114 ± 21 bpm; p < 0.001), there was no correlation found between the MPG and the HR, or any other parameter of interest, at either time-point. In a further analysis, a moderate to strong correlation was observed in the linear relationship between the CI and the MPG (r = 0.60; p < 0.001). During the in-hospital follow-up period, no patients died or required an intervention due to LAVV stenosis. CONCLUSIONS: The Doppler-based quantification of diastolic transvalvular LAVV mean pressure gradients using intraoperative transesophageal echocardiography seems to be prone to overestimation due to altered hemodynamics immediately after an AVSD repair. Thus, the current hemodynamic state should be taken into consideration during the intraoperative interpretation of these gradients.

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis.

Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D.

Analysis of Gum proteins involved in xanthan biosynthesis throughout multiple cell fractions in a "single-tube".

Xanthomonas is a phytopathogenic bacterium and of industrial interest due to its capability to produce xanthan, used as a thickener and emulsifier in the food and non-food industry. Until now, proteome analyses of Xcc lacking a detailed view on the proteins involved in xanthan biosynthesis. The proteins involved in the biosynthesis of this polysaccharide are located near, in or at the cell membrane. This study aims to establish a robust and rapid protocol for a comprehensive proteome analysis of Xcc strains, without the need to isolate different cell fractions. Therefore, a method for the analysis of the whole cell proteome was compared to the isolation of specific fractions regarding the total number of identified proteins, the overlap, and the differences between the approaches. The whole cell proteome analysis with extended peptide separation methods resulted in more than 3254 identified proteins covering 73.1% of the whole proteome. The protocol was used to study xanthan production in a label-free quantification approach. Expression profiles of 8 Gum proteins were compared between the stationary and logarithmic growth phase. Differential expression levels within the operon structure indicate a complex regulatory mechanism for xanthan biosynthesis. Data are available via ProteomeXchange with identifier PXD027261. SIGNIFICANCE: Bacteria are metabolite factories with a wide variety of natural products. Thus, proteome analyses play a crucial role to understand the biological processes within a cell behind the biosynthesis of those metabolites. Proteins involved in the biosynthesis of secreted products are often organised on, in or around the membrane allowing metabolite channelling. Experiments targeting those biosynthesis pathways on protein level often require the analysis of multiple cell fractions like cytosolic, inner, and outer membrane. This is time consuming and demands different protocols. The protocol presented here is a rapid and robust solution to study biosynthetic pathways of biological or biotechnological interest in a single approach on protein level, where gene products are partitioned across multiple cell fractions. The use of a single method also simplifies the comparison of different experiments, for example, production vs. nonproduction conditions.

Characterization of Plasmodium falciparum macrophage migration inhibitory factor homologue and its cysteine deficient mutants.

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) is a homologue of the multifunctional human host cytokine MIF (HsMIF). Upon schizont rupture it is released into the human blood stream where it acts as a virulence factor, modulating the host immune system. Whereas for HsMIF a tautomerase, an oxidoreductase, and a nuclease activity have been identified, the latter has not yet been studied for PfMIF. Furthermore, previous studies identified PfMIF as a target for several redox post-translational modifications. Therefore, we analysed the impact of S-glutathionylation and S-nitrosation on the protein's functions. To determine the impact of the four cysteines of PfMIF we produced His-tagged cysteine to alanine mutants of PfMIF via site-directed mutagenesis. Recombinant proteins were analysed via mass spectrometry, and enzymatic assays. Here we show for the first time that PfMIF acts as a DNase of human genomic DNA and that this activity is greater than that shown by HsMIF. Moreover, we observed a significant decrease in the maximum velocity of the DCME tautomerase activity of PfMIF upon alanine replacement of Cys3, and Cys3/Cys4 double mutant. Lastly, using a yeast reporter system, we were able to verify binding of PfMIF to the human chemokine receptors CXCR4, and demonstrate a so-far overlooked binding to CXCR2, both of which function as non-cognate receptors for HsMIF. While S-glutathionylation and S-nitrosation of PfMIF did not impair the tautomerase activity of PfMIF, activation of these receptors was significantly decreased.

The effect of short-term refrigeration on platelet responsiveness.

Storage of platelet concentrates (PC) at cold temperature (CT) is discussed as an alternative to the current standard of storage at room temperature (RT). Recently, we could show that cold-induced attenuation of inhibitory signaling is an important mechanism promoting platelet reactivity. For developing strategies in blood banking, it is required to elucidate the time-dependent onset of facilitated platelet activation. Thus, freshly prepared platelet-rich-plasma (PRP) was stored for 1 and 2 h at CT (2-6 °C) or at RT (20-24 °C), followed by subsequent comparative analysis. Compared to RT, basal and induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation levels were decreased under CT within 1 h by approximately 20%, determined by Western blot analysis and flow cytometry. Concomitantly, ADP- and collagen-induced threshold aggregation values were enhanced by up to 30-40%. Furthermore, platelet-covered areas on collagen-coated slides and aggregate formation under flow conditions were increased after storage at CT, in addition to induced activation markers. In conclusion, a time period of 1-2 h for refrigeration is sufficient to induce an attenuation of inhibitory signaling, accompanied with an enhancement of platelet responsiveness. Short-term refrigeration may be considered as a rational approach to obtain PC with higher functional reactivity for the treatment of hemorrhage.

Sleep restriction prior to antigen exposure does not alter the T cell receptor repertoire but impairs germinal center formation during a T cell-dependent B cell response in murine spleen.

It is well known that sleep promotes immune functions. In line with this, a variety of studies in animal models and humans have shown that sleep restriction following an antigen challenge dampens the immune response on several levels which leads to e.g. worsening of disease outcome and reduction of vaccination efficiency, respectively. However, the inverse scenario with sleep restriction preceding an antigen challenge is only investigated in a few animal models where it has been shown to reduce antigen uptake and presentation as well as pathogen clearance and survival rates. Here, we use injection of sheep red blood cells to investigate the yet unknown effect on a T cell-dependent B cell response in a well-established mouse model. We found that 6 â€‹h of sleep restriction prior to the antigen challenge does not impact the T cell reaction including the T cell receptor repertoire but dampens the development of germinal centers which correlates with reduced antigen-specific antibody titer indicating an impaired B cell response. These changes concerned a functionally more relevant level than those found in the same experimental model with the inverse scenario when sleep restriction followed the antigen challenge. Taken together, our findings showed that the outcome of the T cell-dependent B cell response is indeed impacted by sleep restriction prior to the antigen challenge which highlights the clinical significance of this scenario and the need for further investigations in humans, for example concerning the effect of sleep restriction preceding a vaccination.