Assembly of alternative prothrombinase by extracellular histones initiates and disseminates intravascular coagulation.

Thrombin generation is pivotal to both physiological blood clot formation and pathological development of disseminated intravascular coagulation (DIC). In critical illness, extensive cell damage can release histones into the circulation, which can increase thrombin generation and cause DIC, but the molecular mechanism is not clear. Typically, thrombin is generated by the prothrombinase complex, comprising activated factor X (FXa), activated cofactor V (FVa), and phospholipids to cleave prothrombin in the presence of calcium. In this study, we found that in the presence of extracellular histones, an alternative prothrombinase could form without FVa and phospholipids. Histones directly bind to prothrombin fragment 1 (F1) and fragment 2 (F2) specifically to facilitate FXa cleavage of prothrombin to release active thrombin, unlike FVa, which requires phospholipid surfaces to anchor the classical prothrombinase complex. In vivo, histone infusion into mice induced DIC, which was significantly abrogated when prothrombin F1 + F2 were infused prior to histones, to act as decoy. In a cohort of intensive care unit patients with sepsis (n = 144), circulating histone levels were significantly elevated in patients with DIC. These data suggest that histone-induced alternative prothrombinase without phospholipid anchorage may disseminate intravascular coagulation and reveal a new molecular mechanism of thrombin generation and DIC development. In addition, histones significantly reduced the requirement for FXa in the coagulation cascade to enable clot formation in factor VIII (FVIII)- and FIX-deficient plasma, as well as in FVIII-deficient mice. In summary, this study highlights a novel mechanism in coagulation with therapeutic potential in both targeting systemic coagulation activation and correcting coagulation factor deficiency.

A Cadaveric Study of the Communication Patterns Between the Buccal Trunks of the Facial Nerve and the Infraorbital Nerve in the Midface.

Most nerve communications reported in the literature were found between the terminal branches. This study aimed to clarify and classify patterns of proximal communications between the buccal branches (BN) of the facial nerve and the infraorbital nerve (ION).The superficial musculoaponeurotic system protects any communication sites from conventional dissections. Based on this limitation, the soft tissues of each face were peeled off the facial skull and the facial turn-down flap specimens were dissected from the periosteal view. Dissection was performed in 40 hemifaces to classify the communications in the sublevator space. Communication site was measured from the ala of nose.A double communication was the most common type found in 62.5% of hemifaces. Triple and single communications existed in 25% and 10% of 40 hemiface specimens, respectively. One hemiface had no communication. The most common type of communication occurred between the lower trunk of the BN of the facial nerve and the lateral labial (fourth) branch of the ION (70% in 40 hemifaces). Communication site was deep to the levator labii superioris muscle at 16.2 mm from the nasal ala. Communications between the motor and the sensory nerves in the midface may be important to increase nerve endurance and to compensate functional loss from injury.Proximal communications between the main trunks of the facial nerve and the ION in the midface exist in every face. This implies some specific functions in normal individuals. Awareness of these nerves is essential in surgical procedure in the midface.

DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA.

The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ∼170 kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes.

Ras protein abundance correlates with Ras isoform mutation patterns in cancer.

Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS > NRAS¼HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. We suggest that in most cases, being the most abundant Ras isoform correlates with occupying the sweet-spot and that HRAS and NRAS expression is usually insufficient to promote oncogenesis when mutated. However, our results challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage.

Absolute Quantitation of GTPase Protein Abundance.

Ras proteins and other small molecular weight GTPases are molecular switches controlling a wide range of cellular functions. High homology and functional redundancy between closely related family members are commonly observed. Antibody-based methods are commonly used to characterize their protein expression. However, these approaches are typically semi-quantitative, and the requirement to use different antibodies means that this strategy is not suited for comparative analysis of the relative expression of proteins expressed by different genes. We present a mass spectrometry-based method that precisely quantifies the protein copy number per cell of a protein of interest. We provide detailed protocols for the generation of isotopically labeled protein standards, cell/tissue processing, mass-spectrometry optimization, and subsequent utilization for the absolute quantitation of the abundance of a protein of interest. As examples, we provide instructions for the quantification of HRAS, KRAS4A, KRAS4B, NRAS, RALA, and RALB in cell line and tissue-derived samples.

Anatomical and ultrasound-based injections for sunken upper eyelid correction.

BACKGROUND: A needle or a cannula can be safely used during filler injection procedures to correct a sunken upper eyelid. To date, there are no precise injection points recommended that are based on an anatomical study. OBJECTIVE: This study systematically investigated the vascular pattern and depth of forehead arteries at the periorbital area of upper eyelid. METHODS: Twenty cadavers were dissected in this study. Additional data were obtained from 30 healthy volunteers using Doppler ultrasound imaging with high-frequency probe. RESULTS: The ophthalmic artery divided into two opposite primary branches: the superior and inferior orbitoglabellar arteries running along the orbital rim. After the supratrochlear artery arose from the superior orbitoglabellar artery at the medial eyebrow, the supraorbital artery either divided from this artery near the supraorbital foramen or emerged as an individual artery from the supraorbital notch. The inferior orbitoglabellar artery gave off the radix artery superior to the medial canthal tendon. The radix artery divided into two opposite branches: the dorsal nasal artery going to the nose and the paracentral artery going to the glabella. Ultrasound imaging revealed a subcorrugator space that a cannula can safely pass through. At the supraorbital foramen/notch, the supraorbital artery traveled very close to the bone. Based on the anatomical data collected, the following injection points for a needle and a cannula technique are recommended. CONCLUSION: Correction of a sunken upper eyelid is a dangerous procedure which should be performed only by experienced physicians. However, with precise anatomical knowledge and correct techniques, optimal outcomes can be safely achieved.

Investigation of the presence and variation of the ascending mental artery: Conventional dissections and ultrasonographic study.

BACKGROUND: Tongue and mouth floor infarction following filler injections for chin augmentation is a rare complication that has the increase in incidence been reported. OBJECTIVE: This study investigated the arterial anastomosis between the submental and sublingual arteries that can lead to the emboli and subsequent tongue infarction during chin augmentation. METHODS: Forty-two formaldehyde-embalmed cadavers and four soft-embalmed cadavers were dissected to verify the incidence and source of the ascending mental artery. Ultrasonographic study of the artery was performed in 10 healthy volunteers. Attention was paid to discriminate whether the ascending mental artery arose from the submental artery or the sublingual artery using the arch of the mylohyoid muscle as the discriminating landmark. RESULTS: Incidence of ascending mental artery from the sublingual artery was 7.1% in the studied population. All ascending mental arteries were 0.7 ± 0.2 mm in diameter at the mental protuberance and were branches of the submental artery that arose from the facial artery, except for two arteries that arose from the sublingual artery. Ultrasonographic study revealed that one left and one right sublingual artery from the lingual arteries penetrated the mylohyoid muscle near the midline to become the ascending mental artery in two volunteers. The ascending mental artery from the other side continued from the submental artery. CONCLUSION: Findings from the cadaveric dissections and ultrasonographic study revealed that the ascending mental artery may be a branch that continues from the lingual artery, or communicates with the sublingual artery through the mouth floor.

Ultrasound evaluation of arterial anastomosis of the forehead.

BACKGROUND: Color Doppler ultrasound has a potential role as an imaging guide in aiding filler injections which are blinded procedures. OBJECTIVE: This study investigated the forehead arteries and provided insight into their anastomoses. This was performed by challenging their function to provide blood through these anastomoses when the main artery was temporary occluded by compression. METHODS: Three arteries were identified on each side of the forehead, the supratrochlear, the supraorbital and the superficial temporal arteries. Under ultrasound monitoring, each target artery and corresponding anastomosis was studied separately by compressions performed in a sequential and accumulative manner. RESULTS: Data from the current study imply that accidental cannulation of either the supratrochlear artery or the supraorbital artery can cause ophthalmic artery embolization in every case recorded. If the frontal branch of the superficial temporal artery is cannulated, the chance of blindness as a complication occurs in one fifth of volunteers. Anastomosis between both sides of the terminal branches of ophthalmic arteries creates the possibility of bilateral ocular complications when accidental cannulation occurs at one of these branches, especially the supratrochlear artery. Thus, injury to the supratrochlear artery carries a greater risk of complication than the supraorbital artery. CONCLUSION: These findings emphasize that the chance of ocular complication is less when accidental cannulation occurs at the superficial temporal artery compared with injury to the supratrochlear or the supraorbital arteries as the terminal branches of the ophthalmic artery. Ultrasound can assist in the identification and evaluation of all the arteries at risk, thus avoiding the occurrence of vascular complications.

Isolation of nuclei in media containing an inert polymer to mimic the crowded cytoplasm.

Within cells, the nucleus is surrounded by the cytoplasm which contains diffusible macromolecules at a high concentration (>100 mg/ml). When cells are broken to isolate nuclei by current methods these macromolecules are dispersed, and to reproduce the environment of nuclei in vivo more closely we have developed a method to isolate them in a medium where cytoplasmic macromolecules are replaced by an inert, volume-occupying polymer and which is essentially cation-free. Nuclei isolated by this method resemble closely those prepared by conventional procedures as seen by optical and electron microscopy, and their internal compartments (nucleoli, PML and Cajal bodies, transcription centers, and splicing speckles) and transcriptional activity are conserved. This procedure is efficient for mammalian cells that normally grow in suspension and do not have an extensive cytoskeleton, and requires ~30 min.

Non-specificity of Pitstop 2 in clathrin-mediated endocytosis.

Small molecule inhibitors of clathrin-mediated endocytosis are highly desired for the dissection of membrane trafficking pathways in the lab and for potential use as anti-infectives in the clinic. One inhibition strategy is to prevent clathrin from contacting adaptor proteins so that clathrin-mediated endocytosis cannot occur. "Pitstop" compounds have been developed that block only one of the four functional interaction sites on the N-terminal domain of clathrin heavy chain. Despite this limitation, Pitstop 2 causes profound inhibition of clathrin-mediated endocytosis. In this study, we probed for non-specific activity of Pitstop 2 by examining its action in cells expressing clathrin heavy chain harbouring mutations in the N-terminal domain interaction sites. We conclude that the inhibition observed with this compound is due to non-specificity, i.e. it causes inhibition away from its proposed mode of action. We recommend that these compounds be used with caution in cells and that they should not be used to conclude anything of the function of clathrin's N-terminal domain.

Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small-scale motion and irregular conformation of chromatin seen in vivo are not reproduced in nuclei isolated in conventional ionic media.