Catheter-related thrombosis in children with intestinal failure and long-term parenteral nutrition: How to treat and to prevent?

Survival of children with chronic intestinal failure has increased as result of administration of home parenteral nutrition. Crucial for the successful management of home parenteral nutrition is the availability of an adequate central venous access. Venous access can be fraught by episodes of catheter-infection, recurrent replacements of catheters and finally catheter-related thrombosis. Management and prevention of catheter-related thrombosis are of vital importance. For patients with compromised venous access, alternative measures are reported, most of them used as a bridge to bowel transplantation.     

Tyrosine phosphorylation-dependent localization of TmaR that controls activity of a major bacterial sugar regulator by polar sequestration

The poles of Escherichia coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a previously unannotated protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization and activity of the general PTS protein Enzyme I (EI) by binding and polar sequestration of EI, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably, phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results suggest that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first, to our knowledge, E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.

The central dogma describes the flow of genetic information from DNA to RNA to protein. However, for this process to be successful, the final product—the protein—needs to be in the right place and at the right time. The consequences of mislocalization can be harmful to any cell type, let alone to the unicellular bacterial cell, whose survival depends on fast and efficient response to environmental changes. Hence, protein localization is an important posttranslational regulatory step. Thus far, most examples of protein targeting were reported in eukaryotic cells, usually in the context of transport from one organelle to another (1). In recent years, it became evident that localization of proteins and RNAs to specific subcellular domains occurs also in prokaryotic cells and is vital for many cellular processes (2–5). However, the mechanisms underlying macromolecules targeting to specific subcellular domains in bacterial cells, with the exception of membrane and cell division proteins, remain largely unknown.The bacterial cell poles are emerging as important domains that accommodate protein and RNA assemblies (5, 6). Pole-localized proteins are involved in a wide range of cellular functions, including motility, regulation of cell cycle, metabolism, differentiation, pathogenesis, and secretion (7). Several proteins were reported to be kept as inactive at the Escherichia coli cell pole until needed, e.g., MurG (8), and FtsZ (9). The phosphotransferase system (PTS), which controls sugar utilization and metabolism in most bacteria, provides an example for regulation via polar cluster formation. Execution of the PTS functions depends on a phosphorylation cascade that initiates with EI and HPr—the general PTS proteins—that deliver the phosphate to the PTS sugar permeases, which import and phosphorylate the incoming sugars (10). The PTS proteins also exert different effects on non-PTS proteins depending on their phosphorylation on histidine residues, thus modulating the hierarchy in sugar utilization (10). The PTS-imported sugars enter glycolysis, whose product, phosphoenolpyruvate (PEP), phosphorylates EI, making EI an important link between glycolysis and sugar uptake (11). We have previously shown that the general PTS proteins localize to the E. coli cell poles (12), although their localization depends on yet-unknown factors (13), that during growth EI polar clusters form stochastically from preexisting dispersed molecules, and that EI clustering negatively correlates with EI function (14). Still, conclusive proof for polar localization as an inhibitory mechanism of EI function is lacking and the identity of the factor that captures EI at the pole remained unknown.Polar clusters offer additional benefits, such as communication between signal transduction systems in order to generate an optimal response, e.g., the chemotaxis and the PTS system (15), or the establishment of cellular asymmetry to coordinate developmental programs with cell cycle progression (7). Polar proteins that recruit other proteins to the poles, thus regulating cell cycle progression, were discovered in some bacteria, e.g., DivIVA in Bacillus subtilis, PopZ and TipN in Caulobacter crescentus, and HubP in Vibrio cholerae (7). In E. coli, the three Min proteins, MinCDE, cooperate to position the cell division site through pole-to-pole oscillation (16).In some bacteria, e.g., C. crescentus, specific localization of proteins is linked to their phosphorylation or to the phosphorylation of factors regulating them (17, 18). In most cases, these proteins are members of the two-component systems, which mediate sensing and regulation by phosphorylation on histidine and aspartic acid residues (19), events considered most prevalent in bacteria. Only in recent years, improved methodologies revealed numerous previously unknown Ser/Thr/Tyr phosphorylation sites, once thought to be hallmarks of eukaryotes, in bacterial and archaeal proteins. The degree to which these putative sites are phosphorylated is still unclear and proofs for their importance in vivo are just beginning to emerge. Also, the linkage between phosphorylation on Ser/Thr/TyR and localization of the phosphorylated bacterial proteins remained unknown.In this study, we show that a previously uncharacterized E. coli protein, YeeX, which is prevalent among Gram-negative bacteria, clusters at the pole in a tyrosine phosphorylation-dependent manner and recruits the major sugar utilization regulator EI. We, therefore, renamed this protein TmaR for targeting of sugar metabolism-associated regulator. TmaR and EI are shown to physically interact and to colocalize. TmaR is necessary for EI polar clustering, but the opposite is not true. Only phosphorylated, TmaR can release EI from the poles, since the diffuse nonphosphorylated TmaR binds to EI quite irreversibly. Notably, tyrosine phosphorylation of EI is also required for its polar localization. We further show that TmaR-mediated EI clustering inversely correlates with EI-mediated sugar uptake, implying that the polar clusters serve as a reservoir for ready-to-act EI molecules. Cells lacking TmaR have detrimental effects on cell survival, which is affected by EI and sugar concentration, when challenged with mildly acidic conditions that are typical to various E. coli habitats. Taken together, our study identifies TmaR as a spatial regulator of sugar metabolism and bacterial survival.     

Possible association of Toll-like receptor 9 polymorphisms with cytokine levels and posttraumatic symptoms in individuals with various types of orthopaedic trauma: Early findings

Background

Although TLR9 polymorphisms may be associated with cytokine dysregulation, its role in regulation of cytokines due to bodily trauma or in relation to acute stress symptoms or posttraumatic stress symptoms (ASS/PTS) has not been evaluated.

Aims

To assess serum cytokine levels and levels of ASS and PTS in relation to four common TLR9 single-nucleotide polymorphisms (SNPs) in individuals with various types of orthopaedic trauma.

Methods

Forty-eight accident-injured individuals, aged 20–60 years were studied. Serum cytokine levels and TLR9 SNPS (1486T/C, 1237T/C, 1174G/A and 2848G/A) were assessed together with intensity of ASS and PTS symptoms.

Results

Statistically significant higher serum levels of IL-12 and IL-1β (p < .05) were found in individuals heterozygous for TLR9-1237 (TC) than in individuals expressing the most common TLR9-1237 type (TT), while differences in levels of IL-6 were not significant. Also, marginally significant levels of IL-6 were found in individuals expressing the common TLR9-1174 (GG) compared with individuals homozygous (AA) or heterozygous (GA) for this SNP. They also had non-significant higher intensity of ASS symptoms. A trend of higher PTS levels in individuals expressing the most common type TLR9-1174 (GG) was found, contrary to homozygous (AA) and heterozygous individuals (GA).

Conclusions

The results of this pilot study suggest that accident-injured individuals with certain TLR9 polymorphisms express higher levels of pro-inflammatory cytokines (IL-1β, IL-6 and IL-12). The associations of TLR9 SNPSs with increased risk of ASS or PTS should be further studied in larger groups of such patients.     

三七三醇皂苷对局灶性脑缺血再灌注大鼠脑组织Nogo-A表达的影响

目的:了解局灶性脑缺血再灌注后Nogo-A的动态变化,以及三七三醇皂苷(PTS)对其表达的影响。方法:建立局灶脑缺血再灌注模型,采用免疫组织化学技术结合Western免疫印迹技术、功能评分,检测72只缺血再灌注后不同时期(3 d,7 d)脑内Nogo-A蛋白的表达并评价功能恢复。结果:Western免疫印迹和免疫组织化学均显示:Nogo-A表达在缺血再灌注损伤后3 d时下降,7 d时上升。PTS组在3 d时的Nogo-A表达比模型组低(P>0.05)。PTS组7 d时Nogo-A表达较模型组低(P<0.05)。结论:PTS可使缺血再灌注大鼠的Nogo-A表达下降,这可能是其发挥脑保护作用的机理之一。     

三七三醇皂苷对脑缺血再灌注大鼠的保护作用

目的 通过对局灶性脑缺血大鼠不同再灌注时段的动态观察．探讨三七三醇皂苷(PTS)对大鼠局灶性脑缺血／再灌注动物模型的神经行为学和脑梗死体积的保护作用。方法 采用改良的线栓法制备大脑中动脉阻塞(MACO)2h、再灌注不同时间段(3h、6h、12h、24h、48h、72h、7d)的大鼠短暂局灶性脑缺血模型。动物随机分假手术组、生理盐水对照组、三七三醇皂苷(PTS)组。用Zea Longa5分制评分和TTC染色法评价神经行为学和脑梗死体积。结果 神经行为学评分除72h组有明显改善外．其余各组与生理盐水对照组比较无显著性差异。脑梗死体积除再灌注3h、6h外．其余各组与生理盐水组比较差异均有显著性意义。结论 三七三醇皂苷对大鼠局灶性脑缺血及再灌注损伤有一定的保护作用。     

Pharmacomechanical Thrombolysis of Symptomatic Acute and Subacute Deep Vein Thrombosis with a Rotational Thrombectomy Device

PurposeTo retrospectively evaluate the efficacy and safety of pharmacomechanical thrombolysis (PMT) with the use of a rotational thrombectomy device for symptomatic deep vein thrombosis (DVT).Materials and MethodsBetween July 2012 and August 2013, 41 patients with acute or subacute DVT underwent PMT. The Cleaner thrombectomy device was used in a single-session technique for patients with lower-extremity DVT. Based on contrast venography, the extent of lysis was graded from I (< 50%) to III (complete).ResultsSixteen patients (39.0%) had a femoropopliteal thrombosis and 25 (61.0%) had an iliofemoral venous thrombosis. The mean duration of symptoms was 11.0 days (range, 3–25 d). The mean quantity of tissue plasminogen activator was 20.7 mg (range, 10–50), and the mean duration of the procedure was 74.3 minutes (range, 30–240 min). At the end of the PMT procedure, 29 patients (70.7%) had complete (grade III) thrombus resolution. Grade I and II lysis were noted in one (2.4%) and 11 (26.8%) patients, respectively. Thirty-eight of the 41 patients were treated with PMT in a single session, and three (7.3%) required an additional lytic infusion as a result of residual thrombi. The overall grade III, II, and I thrombus resolution rates, including the supplemental thrombolysis, were 73.2% (n = 30), 22.0% (n = 9), and 4.9% (n = 2), respectively. There was no mortality.ConclusionsUse of the Cleaner thrombectomy device is a promising alternative to current treatment modalities for the management of DVT in a single session of PMT.     

Production of microbial secondary metabolites: Regulation by the carbon source

Microbial secondary metabolites are low molecular mass products, not essential for growth of the producing cultures, but very important for human health. They include antibiotics, antitumor agents, cholesterol-lowering drugs, and others. They have unusual structures and are usually formed during the late growth phase of the producing microorganisms. Its synthesis can be influenced greatly by manipulating the type and concentration of the nutrients formulating the culture media. Among these nutrients, the effect of the carbon sources has been the subject of continuous studies for both, industry and research groups. Different mechanisms have been described in bacteria and fungi to explain the negative carbon catabolite effects on secondary metabolite production. Their knowledge and manipulation have been useful either for setting fermentation conditions or for strain improvement. During the last years, important advances have been reported on these mechanisms at the biochemical and molecular levels. The aim of the present review is to describe these advances, giving special emphasis to those reported for the genus Streptomyces.