Median arcuate ligament syndrome with retroperitoneal haemorrhage: A case report.

BACKGROUND: Median arcuate ligament syndrome (MALS) is relatively rare and is due to extraluminal compression of the coeliac artery by the median arcuate ligament of the diaphragm. Here, we report a case of MALS found in a patient with abdominal pain and retroperitoneal haemorrhage for education and dissemination. CASE SUMMARY: This article describes a 46-year-old female patient who was admitted to our hospital with abdominal pain as her chief complaint. She had experienced no obvious symptoms but had retroperitoneal bleeding during the course of the disease. Contrast-enhanced computed tomography (CT) and noninvasive CT angiography (CTA) led to an initial misdiagnosis of pancreaticoduodenal artery aneurysm (PDAA) causing retroperitoneal hemorrhage. After intraoperative exploration and detailed analysis of enhanced CT and CTA images, a final diagnosis of MALS was made. The cause of the haemorrhage was bleeding from a branch of the gastroduodenal artery, not rupture of a PDAA. The prognosis of MALS combined with PDAA treated by laparoscopy and interventional therapy is still acceptable. The patient was temporarily treated by gastroduodenal suture haemostasis and was referred for further treatment. CONCLUSION: MALS is very rare and usually has postprandial abdominal pain, upper abdominal murmur, and weight loss. It is diagnosed by imaging or due to complications. When a patient has abdominal bleeding or PDAA, we should consider whether the patient has celiac trunk stenosis (MALS or other etiology). When abdominal bleeding is combined with an aneurysm, we generally think of aneurysm rupture and hemorrhage first, but it may also be collateral artery rupture and hemorrhage.

Intracellular membranes of bacterial endospores are reservoirs for spore core membrane expansion during spore germination.

Bacterial endospores are formed by certain bacteria, such as Bacillus subtilis or the pathogenic Bacillus anthracis and Clostridioides difficile, to allow survival in environmental conditions which are lethal to vegetative bacteria. The spores possess a particular architecture and molecular inventory which endow them with a remarkable resistance against desiccation, heat and radiation. Another remarkable spore feature is their rapid return to vegetative growth during spore germination and outgrowth. The underlying processes of this latter physiological and morphological transformation involve a number of different events, some of which are mechanistically not entirely understood. One of these events is the expansion of the central spore core, which contains the DNA, RNA and most spore enzymes. To date, it has been unclear how the ~1.3- to 1.6-fold expansion of the core membrane surface area that accompanies core expansion takes place, since this occurs in the absence of significant if any ATP synthesis. In the current work, we demonstrate the presence of intracellular membrane structures in spores located just below the core membrane. During spore germination these internal core membranes disappear when the core size increases, suggesting that they are integrated into the core membrane to allow core expansion. These intracellular membranes are most probably present as more or less compressed vesicles or tubules within the dormant spore core. Investigations of spores from different species suggest that these intracellular membrane structures below the core membrane are a general feature of endospore forming bacteria.

Reversible Cryopreservation of Living Cells Using an Electron Microscopy Cryo-Fixation Method.

Rapid cooling of aqueous solutions is a useful approach for two important biological applications: (I) cryopreservation of cells and tissues for long-term storage, and (II) cryofixation for ultrastructural investigations by electron and cryo-electron microscopy. Usually, both approaches are very different in methodology. Here we show that a novel, fast and easy to use cryofixation technique called self-pressurized rapid freezing (SPRF) is-after some adaptations-also a useful and versatile technique for cryopreservation. Sealed metal tubes with high thermal diffusivity containing the samples are plunged into liquid cryogen. Internal pressure builds up reducing ice crystal formation and therefore supporting reversible cryopreservation through vitrification of cells. After rapid rewarming of pressurized samples, viability rates of > 90% can be reached, comparable to best-performing of the established rapid cooling devices tested. In addition, the small SPRF tubes allow for space-saving sample storage and the sealed containers prevent contamination from or into the cryogen during freezing, storage, or thawing.

Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization.

Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth.

The Effect of ADAM8 on the Proliferation and Apoptosis of Hepatocytes and Hepatoma Carcinoma Cells.

This study was undertaken to evaluate the effect of ADAM8 on the proliferation and apoptosis of hepatocytes and hepatoma carcinoma cells during hepatocellular carcinoma (HCC) progression. The expression of ADAM8 was significantly increased with good correlation of PCNA expression increasing and cells apoptosis decreasing during the progression of HCC in the liver of mice. Proliferation experiment in vitro showed that recombinant ADAM8 could induce the expression of PCNA in L02 cells, but not in HepG2 cells. Apoptosis experiment in vitro showed that recombinant ADAM8 did not induce or inhibit the expression of apoptosis-related factors Bcl2, Bax, and Caspase3 in L02 cells, but significantly induced the expression of Bcl2, inhibited the expression of Bax and Caspase3 in HepG2 cells. In conclusion, our study suggested that ADAM8 could promote the proliferation of normal hepatocytes and render hepatoma carcinoma cells more resistant to apoptosis to play important roles during the progression of HCC. ADAM8; Proliferation; Apoptosis.

The Protective Roles of IL-6 Trans-Signaling Regulated by ADAM9 on the Liver in Carbon Tetrachloride-Induced Liver Injury in Mice.

Our study was undertaken to evaluate the important role that a disintegrin and metalloproteinase 9 (ADAM9) regulates IL-6 trans-signaling in carbon tetrachloride (CCl4)-induced liver injury in mice. Mice were divided into four groups. Each group respectively received mineral oil injection, CCl4 injection, anti-ADAM9 monoclonal antibody (mAb) pretreatment and CCl4 injection, anti-ADAM9 mAb and recombinant mouse ADAM9 molecules pretreatment with CCl4 injection. Our results showed that anti-ADAM9 mAb pretreatment significantly aggravated liver injury, inhibited IL-6 trans-signaling, which led to downregulation of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), upregulation of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocytes apoptosis at 24 h after CCl4 injection. Recombinant ADAM9 molecules pretreatment reversed the impact of anti-ADAM9 mAb pretreatment in mice. In conclusion, our study suggested that ADAM9 could regulate the hepatocytes proliferation, apoptosis, angiogenesis, and CYP2E1 expression by activating IL-6 trans-signaling and play important protective roles during CCl4-induced liver injury in mice.

Dormant Bacillus spores protect their DNA in crystalline nucleoids against environmental stress.

Bacterial spores of the genera Bacillus and Clostridium are extremely resistant against desiccation, heat and radiation and involved in the spread and pathogenicity of health relevant species such as Bacillus anthracis (anthrax) or Clostridium botulinum. While the resistance of spores is very well documented, underlying mechanisms are not fully understood. In this study we show, by cryo-electron microscopy of vitreous sections and particular resin thin section electron microscopy, that dormant Bacillus spores possess highly ordered crystalline core structures, which contain the DNA, but only if small acid soluble proteins (SASPs) are present. We found those core structures in spores of all Bacillus species investigated, including spores of anthrax. Similar core structures were detected in Geobacillus and Clostridium species which suggest that highly ordered, at least partially crystalline core regions represent a general feature of bacterial endospores. The crystalline core structures disintegrate in a period during spore germination, when resistance against most stresses is lost. Our results suggest that the DNA is tightly packed into a crystalline nucleoid by binding SASPs, which stabilizes DNA fibrils and protects them against modification. Thus, the crystalline nucleoid seems to be the structural and functional correlate for the remarkable stability of the DNA in bacterial endospores.

IL-6 trans-signaling plays important protective roles in acute liver injury induced by acetaminophen in mice.

Our study was undertaken to evaluate the important role of interleukin-6 (IL-6) trans-signaling in acetaminophen (AAP)-induced liver injury. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL-6 trans-signaling, whereas an IL-6/soluble IL-6 receptor (sIL-6R) fusion protein (hyper-IL-6) mimics IL-6 trans-signaling. Using these tools, we investigated the role of IL-6 trans-signaling in AAP-induced liver injury. Blockade of IL-6 trans-signaling during AAP-induced liver injury remarkably increased the levels of serum aspartate aminotransferase and alanine aminotransferase; lowered the level of serum sIL-6R; aggravated liver injury; inhibited the expression of phosphorylation of STAT3 (pSTAT3), proliferating cell nuclear antigen, vascular endothelial growth factor, and glycogen synthesis; and induced the expression of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocyte apoptosis in the liver of mice. In summary, our study suggested that IL-6 trans-signaling plays important protective roles by regulating the hepatocyte proliferation and apoptosis, angiogenesis, CYP2E1 expression, and glycogen metabolism during AAP-induced liver injury in mice.

Cryo-fixation by self-pressurized rapid freezing.

High-pressure freeze fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in vitreous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy of vitreous sections (CEMOVIS) to explore cellular ultrastructure as close as possible to the native state. Here, we describe the procedure of self-pressurized rapid freezing as fast, easy-to-use, and low-cost freeze fixation method, avoiding the usage of a high-pressure freezing (HPF) apparatus. Cells or small organisms are placed in capillary metal tubes, which are tightly closed and plunged directly into liquid ethane cooled by liquid nitrogen. In parts of the tube, crystalline ice is formed and builds up pressure sufficient for the liquid-glass transition of the remaining specimen. The quality of samples is equivalent to preparations by conventional HPF apparatus, allowing for high-resolution cryo-EM applications or for freeze substitution and plastic embedding.

Iterative antimicrobial candidate selection from informed d-/l-Peptide dimer libraries.

Growing resistance to antibiotics, as well as newly emerging pathogens, stimulate the investigation of antimicrobial peptides (AMPs) as therapeutic agents. Here, we report a new library design concept based on a stochastic distribution of natural AMP amino acid sequences onto half-length synthetic peptides. For these compounds, a non-natural motif of alternating D- and L-backbone stereochemistry of the peptide chain predisposed for ß-helix formation was explored. Synthetic D-/L-peptides with permuted half-length sequences were delineated from a full-length starter sequence and covalently recombined to create two-dimensional compound arrays for antibacterial screening. Using the natural AMP magainin as a seed sequence, we identified and iteratively optimized hit compounds showing high antimicrobial activity against Gram-positive and Gram-negative bacteria with low hemolytic activity. Cryo-electron microscopy characterized the membrane-associated mechanism of action of the new D-/L-peptide antibiotics.

Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens.

Volume microscopy at high resolution is increasingly required to better understand cellular functions in the context of three-dimensional assemblies. Focused ion beam (FIB) milling for serial block face imaging in the scanning electron microscope (SEM) is an efficient and fast method to generate such volume data for 3D analysis. Here, we apply this technique at cryo-conditions to image fully hydrated frozen specimen of mouse optic nerves and Bacillus subtilis spores obtained by high-pressure freezing (HPF). We established imaging conditions to directly visualize the ultrastructure in the block face at -150 °C by using an in-lens secondary electron (SE) detector. By serial sectioning with a focused ion beam and block face imaging of the optic nerve we obtained a volume as large as X=7.72 µm, Y=5.79 µm and Z=3.81 µm with a lateral pixel size of 7.5 nm and a slice thickness of 30 nm in Z. The intrinsic contrast of membranes was sufficient to distinguish structures like Golgi cisternae, vesicles, endoplasmic reticulum and cristae within mitochondria and allowed for a three-dimensional reconstruction of different types of mitochondria within an oligodendrocyte and an astrocytic process. Applying this technique to dormant B. subtilis spores we obtained volumes containing numerous spores and discovered a bright signal in the core, which cannot be related to any known structure so far. In summary, we describe the use of cryo FIB-SEM as a tool for direct and fast 3D cryo-imaging of large native frozen samples including tissues.

Golgi apparatus analyzed by cryo-electron microscopy.

In 1898, the Golgi apparatus was discovered by light microscopy, and since the 1950s, the ultrastructure composition is known by electron microscopic investigation. The complex three-dimensional morphology fascinated researchers and was sometimes even the driving force to develop novel visualization techniques. However, the highly dynamic membrane systems of Golgi apparatus are delicate and prone to fixation artifacts. Therefore, the understanding of Golgi morphology and its function has been improved significantly with the development of better preparation methods. Nowadays, cryo-fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in amorphous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy and tomography. Even though the overall morphology of vitrified Golgi stacks is comparable to well-prepared and resin-embedded samples, previously unknown structural details can be observed solely based on their native density. At this point, any further improvement of sample preparation would gain novel insights, perhaps not in terms of general morphology, but on fine structural details of this dynamic organelle.

Regulation of signaling at regions of cell-cell contact by endoplasmic reticulum-bound protein-tyrosine phosphatase 1B.

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B.

Choline inhibits angiotensin II-induced cardiac hypertrophy by intracellular calcium signal and p38 MAPK pathway.

Choline, an agonist of M(3) muscarinic acetylcholine receptors, is a precursor and metabolite of acetylcholine and is also a functional modulator of cellular membrane. However, the effect of choline on cardiac hypertrophy is not fully understood. The present study was therefore designed to explore whether choline could prevent cardiac hypertrophy induced by angiotensin II (Ang II) and clarify its potential mechanisms. Cardiac hypertrophy was induced by 0.6 mg kg(-1) day(-1) Ang II for 2 weeks in the presence or absence of choline pretreatment, while cardiomyocyte hypertrophy was induced by Ang II 0.1 µM for 48 h. We found that choline pretreatment attenuated the increment cell size of cardiomyocytes induced by Ang II both in vivo and in vitro. The high ANP and ß-MHC levels induced by Ang II were also reversed by choline in cardiomyocytes. Meanwhile, choline pretreatment prevented the augment of reactive oxygen species (ROS) and intracellular calcium concentration in Ang II-treated cardiomyocytes. Furthermore, the upregulated phospho-p38 mitogen-activated protein kinase (MAPK) and calcineurin levels by Ang II in ventricular myocytes were attenuated by choline. In conclusion, choline prevents Ang II-induced cardiac hypertrophy through inhibition of ROS-mediated p38 MAPK activation as well as regulation of Ca(2+)-mediated calcineurin signal transduction pathway. Our results provide new insights into the pharmacological role of choline in cardiovascular diseases.

Self-pressurized rapid freezing (SPRF) as a simple fixation method for cryo-electron microscopy of vitreous sections.

Cryo-electron microscopy of vitreous sections (CEMOVIS) is currently considered the method of choice to explore cellular ultrastructure at high resolution as close as possible to their native state. Here, we apply a novel, easy-to-use and low-cost freeze fixation method for CEMOVIS, avoiding the use of high-pressure freezing apparatus. Cells are placed in capillary metal tubes, which are tightly closed and plunged directly into liquid ethane cooled by liquid nitrogen. In some parts of the tube, crystalline ice is formed, building up pressure sufficient for the liquid-glass transition of the remaining specimen. We verified the presence of vitreous ice in these preparations using CEMOVIS and electron diffraction. Furthermore, different tube materials being less poisonous than copper were established to minimize physiological alterations of the specimen. Bacteria, yeast and mammalian cells were tested for molecular resolution. The quality of results is equivalent to samples prepared by conventional high pressure freezing apparatus, thus establishing this novel method as fast, easy-to-use and low-cost freeze fixation alternative for cryo-EM.

Activation of cardiac M3 muscarinic acetylcholine receptors has cardioprotective effects against ischaemia-induced arrhythmias.

Increasing evidence indicates the important roles of M(3) muscarinic acetylcholine receptors (M(3) mAChR) in the regulation and maintenance of cardiac function and heart disease. In the present study, we investigated whether the M(3) mAChR mediates the cardioprotection against ischaemia-induced arrhythmias and the mechanisms involved. Myocardial ischaemia was established in Wistar rats by occlusion of the left anterior descending coronary artery. Rats were treated with choline chloride (an M(3) mAChR agonist; 10 mg/kg, i.v.) 10 min before occlusion. In addition, 4-diphenylacetoxy-N-methylpiperidine-methiodide (4-DAMP; 0.12 µg/kg, i.v.) was administered 5 min before choline in the 4-DAMP-treated group. Ischaemia-induced arrhythmias were evaluated in each group for a period of 1 h after occlusion. After 24 h occlusion, protein and mRNA levels of L-type Ca(2+) channels and the Na(+) /Ca(2+) exchanger (NCX) were determined. Ischaemia-induced arrhythmias following coronary artery occlusion were diminished by choline and this effect was reversed in the 4-DAMP-treated group. In vitro, the effects of myocardial ischaemia were simulated by incubating isolated ventricular cardiomyocytes with Tyrode's solution (pH 6.8). Intracellular Ca(2+) overload was confirmed and this was decreased by choline. Furthermore, choline reduced the L-type Ca(2+) current (I(C) (a,) (L) ) compared with cardiomyocytes incubated in Tyrode's solution (pH 6.8) alone. Choline reduced the 'ischaemia'-induced upregulated expression of L-type Ca(2+) channels and NCX at both the protein and mRNA level. Based on these results, choline has an obvious protective effect against ischaemia-induced arrhythmias that is mediated via activation of cardiac M(3) mAChR, which reduces Ca(2+) overload mediated by L-type Ca(2+) channels and the NCX.

[Role of traditional Chinese medicine injection infusion with precise infusion apparatus].

The adverse reactions caused by the traditional Chinese medicine has become increasingly aroused great attention. Traditional Chinese medicine injection may produce insoluble particles in the process of production, preparation and infusion, which is one of the reasons caused some adverse drug reactions such as phlebitis, allergic reactions, so it is essential that how to control Chinese medicine injection of insoluble drug particles enter human clinical safety. The author retrieve a large number of paper, from the infusion of particles, analysis traditional Chinese medicine injection and insoluble particles in the liquid compatibility increased occurrence and cause, comparative advantages and disadvantages of the ordinary infusion and precision infusion, which found that using sophisticated filters infusion of traditional Chinese medicine injection control of traditional Chinese medicine injection of insoluble particles enter the blood vessels, thereby reducing the occurrence of adverse drug reactions part of the traditional Chinese medicine injection, is the best, most effective, most secure way of infusion.

The antiarrhythmic effect and possible ionic mechanisms of pilocarpine on animal models.

This study was designed to evaluate the effects of pilocarpine and explore the underlying ionic mechanism, using both aconitine-induced rat and ouabain-induced guinea pig arrhythmia models. Confocal microscopy was used to measure intracellular free-calcium concentrations ([Ca(2+)](i)) in isolated myocytes. The current data showed that pilocarpine significantly delayed onset of arrhythmias, decreased the time course of ventricular tachycardia and fibrillation, reduced arrhythmia score, and increased the survival time of arrhythmic rats and guinea pigs. [Ca(2+)](i) overload induced by aconitine or ouabain was reduced in isolated myocytes pretreated with pilocarpine. Moreover, M(3)-muscarinic acetylcholine receptor (mAChR) antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) partially abolished the beneficial effects of pilocarpine. These data suggest that pilocarpine produced antiarrhythmic actions on arrhythmic rat and guinea pig models induced by aconitine or ouabain via stimulating the cardiac M(3)-mAChR. The mechanism may be related to the improvement of Ca(2+) handling.

[A multicenter prospective cohort study on risk factors for hospital-acquired pneumonia in the elderly].

OBJECTIVE: To investigate the incidence and the risk factors for hospital-acquired pneumonia (HAP) in the elderly in Shanghai. METHODS: This was a multicenter prospective clinical cohort study. A total of 5299 patients more than 65 years old, admitted into 31 secondary or tertiary hospitals in Shanghai, were enrolled. Measurements of the demographic and potential risk factors reflecting illness severity, nutrition, drug exposure, surgery and ventilation were performed. Pneumonia was classified by the definition of Chinese Medical Association. Risk factors were analyzed by univariate Pearson Chi-squared test and multivariable logistic regression analysis with backward (Likelihood ratio). RESULTS: Of the enrolled patients, 2805 male and 2494 female, 255 (4.81%) developed hospital-acquired pneumonia. The incidence was 46.75/1000 hospitalizations. Among them 38 died; and the rough mortality was 14.90%. The incidence of HAP was higher in ICU (21.43%), hematology (12.17%), chest surgery (11.41%), and respiratory medicine (7.92%) departments. The mean of acute physiology and chronic health evaluation (APACHE II) score was 8.3 +/- 3.4 (5 - 31). Multivariable logistic regression analysis with backward (Wald) method found that admission into secondary hospitals, admission into ICU, history of chronic obstructive pulmonary disease > or = 10 years, immunosuppression, administration of antibiotics, insertion of nasogastric tube, mechanical ventilation, administration of H-2 antagonists or antacid and < or = 7 d, central nervous system diseases, depressed level of consciousness, supine position, albumin < 35 g/L were independent risk factors of HAP in the elderly. CONCLUSION: Hospital-acquired pneumonia in the elderly was the usual type of nosocomial infections. The risk factors identified from this study may prove useful to target future clinical interventions to prevent HAP in the elderly.

Structural and functional analysis of natrin, a venom protein that targets various ion channels.

Cysteine-rich secretory proteins (CRISPs) are secreted single-chain proteins found in different sources. Natrin is a member of the CRISP family purified from the snake venom of Naja naja atra, which has been reported as a BKca channel blocker. In our study, crystals of natrin were obtained in two different crystal forms and the structure of one of them was solved at a resolution of 1.68A. Our electrophysiological experiments indicated that natrin can block the ion channel currents of the voltage-gated potassium channel Kv1.3. Docking analyses of the interaction between natrin and Kv1.3 revealed a novel interaction pattern different from the two previously reported K(+) channel inhibition models termed "functional dyad" and "basic ring". These findings offered new insights into the function of natrin and how the specific interactions between CRISPs and different ion channels can be achieved.