Cryo-EM structure and biochemical analyses of the nucleosome containing the cancer-associated histone H3 mutation E97K.

The Lys mutation of the canonical histone H3.1 Glu97 residue (H3E97K) is found in cancer cells. Previous biochemical analyses revealed that the nucleosome containing the H3E97K mutation is extremely unstable as compared to the wild-type nucleosome. However, the mechanism by which the H3E97K mutation causes nucleosome instability has not been clarified yet. In the present study, the cryo-electron microscopy structure of the nucleosome containing the H3E97K mutation revealed that the entry/exit DNA regions of the H3E97K nucleosome are disordered, probably by detachment of the nucleosomal DNA from the H3 N-terminal regions. This may change the intra-molecular amino acid interactions with the replaced H3 Lys97 residue, inducing structural distortion around the mutated position in the nucleosome. Consistent with the nucleosomal DNA end flexibility and the nucleosome instability, the H3E97K mutation exhibited reduced binding of linker histone H1 to the nucleosome, defective activation of PRC2 (the essential methyltransferase for facultative heterochromatin formation) with a poly-nucleosome, and enhanced nucleosome transcription by RNA polymerase II.

Cryo-EM and biochemical analyses of the nucleosome containing the human histone H3 variant H3.8.

Histone H3.8 is a non-allelic human histone H3 variant derived from H3.3. H3.8 reportedly forms an unstable nucleosome, but its structure and biochemical characteristics have not been revealed yet. In the present study, we reconstituted the nucleosome containing H3.8. Consistent with previous results, the H3.8 nucleosome is thermally unstable as compared to the H3.3 nucleosome. The entry/exit DNA regions of the H3.8 nucleosome are more accessible to micrococcal nuclease than those of the H3.3 nucleosome. Nucleosome transcription assays revealed that the RNA polymerase II (RNAPII) pausing around the superhelical location (SHL) -1 position, which is about 60 base pairs from the nucleosomal DNA entry site, is drastically alleviated. On the other hand, the RNAPII pausing around the SHL(-5) position, which is about 20 base pairs from the nucleosomal DNA entry site, is substantially increased. The cryo-electron microscopy structure of the H3.8 nucleosome explains the mechanisms of the enhanced accessibility of the entry/exit DNA regions, reduced thermal stability and altered RNAPII transcription profile.

Structural and biochemical analyses of the nucleosome containing Komagataella pastoris histones.

Komagataella pastoris is a methylotrophic yeast that is commonly used as a host cell for protein production. In the present study, we reconstituted the nucleosome with K. pastoris histones and determined the structure of the nucleosome core particle by cryogenic electron microscopy. In the K. pastoris nucleosome, the histones form an octamer and the DNA is left-handedly wrapped around it. Micrococcal nuclease assays revealed that the DNA ends of the K. pastoris nucleosome are somewhat more accessible, as compared with those of the human nucleosome. In vitro transcription assays demonstrated that the K. pastoris nucleosome is transcribed by the K. pastoris RNA polymerase II (RNAPII) more efficiently than the human nucleosome, while the RNAPII pausing positions of the K. pastoris nucleosome are the same as those of the human nucleosome. These results suggested that the DNA end flexibility may enhance the transcription efficiency in the nucleosome but minimally affect the nucleosomal pausing positions of RNAPII.

Unusual nucleosome formation and transcriptome influence by the histone H3mm18 variant.

Histone H3mm18 is a non-allelic H3 variant expressed in skeletal muscle and brain in mice. However, its function has remained enigmatic. We found that H3mm18 is incorporated into chromatin in cells with low efficiency, as compared to H3.3. We determined the structures of the nucleosome core particle (NCP) containing H3mm18 by cryo-electron microscopy, which revealed that the entry/exit DNA regions are drastically disordered in the H3mm18 NCP. Consistently, the H3mm18 NCP is substantially unstable in vitro. The forced expression of H3mm18 in mouse myoblast C2C12 cells markedly suppressed muscle differentiation. A transcriptome analysis revealed that the forced expression of H3mm18 affected the expression of multiple genes, and suppressed a group of genes involved in muscle development. These results suggest a novel gene expression regulation system in which the chromatin landscape is altered by the formation of unusual nucleosomes with a histone variant, H3mm18, and provide important insight into understanding transcription regulation by chromatin.

Structural basis for the inhibition of cGAS by nucleosomes.

The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) senses invasion of pathogenic DNA and stimulates inflammatory signaling, autophagy, and apoptosis. Organization of host DNA into nucleosomes was proposed to limit cGAS autoinduction, but the underlying mechanism was unknown. Here, we report the structural basis for this inhibition. In the cryo-electron microscopy structure of the human cGAS-nucleosome core particle (NCP) complex, two cGAS monomers bridge two NCPs by binding the acidic patch of the histone H2A-H2B dimer and nucleosomal DNA. In this configuration, all three known cGAS DNA binding sites, required for cGAS activation, are repurposed or become inaccessible, and cGAS dimerization, another prerequisite for activation, is inhibited. Mutating key residues linking cGAS and the acidic patch alleviates nucleosomal inhibition. This study establishes a structural framework for why cGAS is silenced on chromatinized self-DNA.

A Thermal Tactile Sensation Display with Controllable Thermal Conductivity.

We demonstrate a thermal tactile sensation display that can present various thermal sensations, namely cold/cool/warm/hot feelings, by varying the effective thermal conductivity of the display. Thermal sensation is one of the major tactile sensations and needs to be further investigated for advanced virtual reality/augmented reality (VR/AR) systems. Conventional thermal sensation displays present hot/cold sensations by changing the temperature of the display surface, whereas the proposed display is the first one that controls its effective thermal conductivity. The device contains an air cavity and liquid metal that have low and high thermal conductivity, respectively. When the liquid metal is introduced to fill up the air cavity, the apparent thermal conductivity of the device increases. This difference in the thermal conductivity leads to the users experiencing different thermal tactile sensations. Using this device, the threshold to discriminate the effective thermal conductivity was experimentally deduced for the first time. This thermal tactile display can be a good platform for further study of thermal tactile sensation.

[A 65-year-old man with liver metastases after lung cancer resection that responded to concomitant use of gemcitabine and carboplatin].

A 65-year-old male with liver metastases after lung cancer resection was treated with five courses of chemotherapy consisting of gemcitabine (GEM) 1,000 mg/m2 (day 1, 8, every 4 weeks) plus carboplatin (CBDCA) AUC 6 (day 1, every 4 weeks). A partial response (PR) was achieved, his symptoms abated and his quality of life(QOL) improved. Although bone marrow suppression was observed as a side effect, it was within the tolerable range and did not interfere with therapy. This approach may be worth considering as a first-line anti-cancer chemotherapy for recurrence lung cancer.

Preoperative detection of pleural adhesions by chest ultrasonography.

BACKGROUND: The presence of pleural adhesions may render video-assisted thoracoscopic surgery difficult or impossible. The aim of this study was to assess the value of chest ultrasonography in the detection of pleural adhesions prior to thoracotomy. METHODS: Between October 2001 and September 2002, 42 consecutive patients undergoing thoracotomies (including video-assisted thoracic surgery) were evaluated with chest ultrasonography. These patients underwent a preoperative ultrasonic examination of the chest wall using a 7-MHz linear ultrasound probe at 7 points along the chest wall. We measured the movement of the visceral pleural slide. RESULTS: When restricted viscera sliding was defined as less than 1 cm of excursion at the upper thoracic wall during exaggerated respirations, ultrasonography demonstrated a sensitivity of 63.6%, a specificity of 79.4%, a negative predictive value of 87.7%, a positive predictive value of 50.0%, and an overall accuracy of 75.6%. When restricted viscera sliding was defined as less than 2 cm of excursion at the lower thoracic wall during exaggerated respirations, ultrasonography demonstrated a sensitivity of 81.5%, a specificity of 81.0%, a negative predictive value of 96.0%, a positive predictive value of 44.0%, and an overall accuracy of 81.0%. CONCLUSIONS: Chest ultrasonography is moderately accurate in detecting the presence and location of pleural adhesions. Use of preoperative chest sonographic findings to plan trocar placement and to determine the need for an open approach is valuable in helping prevent visceral injury and facilitating video-assisted thoracoscopic surgery.

Triangle target principle for the placement of trocars during video-assisted thoracic surgery.

OBJECTIVE: The baseball-diamond principle is generally used for trocar placement during video-assisted thoracic surgery; however, we are unable to treat all peripheral lung lesions using this principle. Therefore, we have developed another method for determining trocar placement based on a modification of the conventional principle. We have termed this method the triangle target principle. This report describes the instrument positioning that we now use for many video-assisted thoracic surgical procedures. METHODS: We position 3 trocars in an equilateral triangle, with the target lesion at the apex. One vertex of the base becomes the site of the first trocar placement for introduction of the thoracoscopic camera. Another vertex of the base becomes the site for the second trocar for forceps or the endoscopic stapler. The third trocar is for forceps and is inserted to create the vicinity of target lesion. Four types of the triangle target principle were developed according to sites of the target lesion. RESULTS: Between January 2000 and December 2002, we used this principle for 161 patients who underwent video-assisted thoracic surgery and all intrathoracic lesions were accessible except in 3 patients requiring intraoperative modifications. CONCLUSIONS: We conclude that video-assisted thoracic surgery by this principle is more effective and easier than the conventional principle to treat intrathoracic disease.

Giant mediastinal bronchial artery aneurysm mimicking benign esophageal tumor: a case report and review of 26 cases from literature.

Mediastinal bronchial artery aneurysm is rare but potentially life-threatening, and requires prompt treatment to avert rupture with catastrophic results. A 78-year-old man was referred to our hospital with a benign esophageal tumor, which appeared as an extrinsic, extramucosal filling defect on an esophagogram. Chest computed tomography and selective bronchial arteriography led to a definitive diagnosis of mediastinal bronchial artery aneurysm. Aneurysmectomy and closure of the ostia of both the afferent and efferent bronchial arteries was performed via standard posterolateral thoracotomy. Postoperative course was uneventful, and the patient was discharged on the seventh postoperative day.

Planning and drawing of occluded objects by young children.

When children under 8 yr. old draw an object occluded by another, they depict each object separately either horizontally or vertically. This study examined whether children aged 4-6-yr.-old intended to draw occluding and occluded objects separately at the planning stage like their drawn product. 25 4-yr.-olds (boys=14, girls=11), 28 5-yr.-olds (boys=14, girls=14), and 29 6-yr.-olds (boys=19, girls=10) were presented two cups, set one behind another, with handles visible at the side in partial occlusion and invisible in total occlusion. They were asked to draw the cups as they saw them and to select one among five picture cards (circle, horizontal, orientation-centered, vertical, view-specific) that matched their drawing plan. Analysis showed in both partial and total occlusions in the drawing 4-yr.-olds more often drew either a circle or the two cups horizontally; 5-yr.-olds drew the two cups vertically, while 6-yr.-olds made view-specific drawings, that is, drew the cups as seen. On the planning task for partial and total occlusions 4-yr.-olds selected the picture card representing a circle or two horizontally drawn cups, 5-yr.-olds tended to pick the card of two vertically drawn cups, while 6-yr.-olds chose the view-specific picture card, like their drawn products. These results suggested that planning and drawing were similar within and across age groups and that children's drawing reflected the representation of their planning.