CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

Open left diaphragm method enables safe surgery with a good visual field in a laparoscopic transhiatal approach for esophagogastric junction adenocarcinoma laparoscopic transhiatal reconstruction via an open left diaphragm method.

BACKGROUND: Despite being oncologically acceptable for esophagogastric junction adenocarcinoma with an esophageal invasion length of 3-4 cm, the transhiatal approach has not yet become a standard method given the difficulty of reconstruction in a narrow space and the risk of severe anastomotic leakage. This study aimed to clarify the safety and feasibility of the open left diaphragm method during the transhiatal approach for esophagogastric junction adenocarcinoma. METHODS: This retrospective study compared the clinical outcomes of patients who underwent proximal or total gastrectomy with lower esophagectomy for Siewert type II/III adenocarcinomas with esophageal invasion via the laparoscopic transhiatal approach with or without the open left diaphragm method from April 2013 to December 2021. RESULTS: Overall, 42 and 13 patients did and did not undergo surgery with the open left diaphragm method, respectively. The median operative time was only slightly shorter in the open left diaphragm group than in the non-open left diaphragm group (369 vs. 482 min; P = 0.07). Grade ≥ II postoperative respiratory complications were significantly less common in the open left diaphragm group than in the non-open left diaphragm group (17% vs. 46%, P = 0.03). Neither group had grade ≥ IV anastomotic leakage, and two cases of anastomotic leakage requiring reoperation were drained using the left diaphragmatic release technique. CONCLUSIONS: Transhiatal lower esophagectomy with gastrectomy using the open left diaphragm method is safe, highlighting its advantages for Siewert type II/III esophagogastric junction adenocarcinoma with an esophageal invasion length of ≤ 4 cm.

A non-randomized, controlled, interventional study to investigate the effects of community pharmacists' cognitive behavioral therapy-based interventions on medication adherence and relevant indicators in patients with depression.

BACKGROUND: The prevalence of depression is increasing in Japan. Pharmacists play an important role in helping patients use medicines effectively. Several studies had investigated the impact of community pharmacists on patient adherence to antidepressant therapy, and their results indicated that further study was warranted. METHODS: This study was conducted from June 2019 to May 2020 using a cluster non-randomized, open-label, parallel-group design. Four community pharmacy stores in Osaka and Hyogo Prefectures, Japan, participated in the study, and enrolled patients with unipolar depression. In the intervention group (IG), patients received cognitive behavioral therapy (CBT)-based medication support, and their medication adherence and adverse drug reactions were monitored by telephone. In the control group (CG), the pharmacists engaged in routine interactions with the study participants. Before participating in this study, the intervention-group pharmacists attended a 5-hour training session on CBT-based medication support. The primary outcome of this study was medication adherence, assessed using the Drug Attitude Inventory (DAI)-10. Secondary outcomes included the changes from baseline at 6 months in the following variables: the Patient Health Questionnaire (PHQ)-9 total score, the EQ-5D-5 L (Euro-QOL 5 dimensions 5 levels) score, patient satisfaction, and the Pharmacists' Confidence Scale about Medication Consultation for Depressive Patients (PCMCD) score. RESULTS: Four pharmacies (two in IG and two in CG) completed the intervention period. Results were obtained from 19 patients in the IG and 12 patients in the CG. In the IG, the mean DAI-10 score increased from 4.941 at baseline to 6.105, the mean PHQ-9 score decreased from 9.263 to 8.625, and the mean patient satisfaction score increased from 39.947 to 42.211. In the CG, the mean DAI-10 score decreased from 6.333 to 4.167, the mean PHQ-9 score increased from 9.333 to 12.923, and the mean patient satisfaction score decreased from 38.929 to 38.167. CONCLUSION: CBT-based medication support provided by community pharmacists may improve patient medication adherence to antidepressant therapy and symptoms. Such support can be expected to facilitate better treatment of depressed patients and may also allow the duration of treatment to be shortened. TRIAL REGISTRATION: UMIN000037954, Date of first registration: 17/06/2019.

Trimethylguanosine synthase 1 (Tgs1) is involved in Swi6/HP1-independent siRNA production and establishment of heterochromatin in fission yeast.

In fission yeast, siRNA generated by RNA interference (RNAi) factors plays critical roles in establishment and maintenance of heterochromatin. To achieve efficient siRNA synthesis, RNAi factors assemble on heterochromatin via association with Swi6, a homologue of heterochromatin protein 1 (HP1), and heterochromatic noncoding RNA (hncRNA) retained on chromatin. In addition, spliceosomes formed on hncRNA introns recruit RNAi factors to hncRNA and heterochromatin. Small nuclear RNAs, components of the spliceosome, have a trimethylguanosine (TMG) cap that is generated by Tgs1-dependent hypermethylation of the normal m7G cap; this cap is required for efficient splicing of some mRNAs in budding yeast and Drosophila. In this study, we found that loss of Tgs1 in fission yeast destabilizes centromeric heterochromatin. Tgs1 was required for Swi6-independent siRNA synthesis, as well as for the establishment of centromeric heterochromatin. Loss of Tgs1 affected the splicing efficiency of hncRNA introns in the absence of Swi6. Furthermore, some hncRNAs have a TMG cap, and we found that loss of Tgs1 diminished the chromatin binding of these hncRNAs. Together, these results suggest that the Tgs1-dependent TMG cap plays critical roles in establishment of heterochromatin by ensuring spliceosome-dependent recruitment of RNAi factors and regulating the binding between chromatin and hncRNA.

Total Synthesis of Loroxanthin.

The first total synthesis of loroxanthin (1) was accomplished by Horner-Wadsworth-Emmons reaction of C25-apocarotenal 8 having a silyl-protected 19-hydroxy moiety with C15-phosphonate 25 bearing a silyl-protected 3-hydroxy-ε-end group. Preparation of apocarotenal 8 was achieved via Stille coupling reaction of alkenyl iodide 10 with alkenyl stananne 9, whereas phosphonate 25 was prepared through treatment of ally alcohol 23 with triethyl phosphite and ZnI2. The ally alcohol 23 was derived from the known (3R,6R)-3-hydroxy C15-aldehyde 20, which was obtained by direct optical resolution of racemate 20 using a semi-preparative chiral HPLC column.

Regulation of ectopic heterochromatin-mediated epigenetic diversification by the JmjC family protein Epe1.

H3K9 methylation (H3K9me) is a conserved marker of heterochromatin, a transcriptionally silent chromatin structure. Knowledge of the mechanisms for regulating heterochromatin distribution is limited. The fission yeast JmjC domain-containing protein Epe1 localizes to heterochromatin mainly through its interaction with Swi6, a homologue of heterochromatin protein 1 (HP1), and directs JmjC-mediated H3K9me demethylation in vivo. Here, we found that loss of epe1 (epe1Δ) induced a red-white variegated phenotype in a red-pigment accumulation background that generated uniform red colonies. Analysis of isolated red and white colonies revealed that silencing of genes involved in pigment accumulation by stochastic ectopic heterochromatin formation led to white colony formation. In addition, genome-wide analysis of red- and white-isolated clones revealed that epe1Δ resulted in a heterogeneous heterochromatin distribution among clones. We found that Epe1 had an N-terminal domain distinct from its JmjC domain, which activated transcription in both fission and budding yeasts. The N-terminal transcriptional activation (NTA) domain was involved in suppression of ectopic heterochromatin-mediated red-white variegation. We introduced a single copy of Epe1 into epe1Δ clones harboring ectopic heterochromatin, and found that Epe1 could reduce H3K9me from ectopic heterochromatin but some of the heterochromatin persisted. This persistence was due to a latent H3K9me source embedded in ectopic heterochromatin. Epe1H297A, a canonical JmjC mutant, suppressed red-white variegation, but entirely failed to remove already-established ectopic heterochromatin, suggesting that Epe1 prevented stochastic de novo deposition of ectopic H3K9me in an NTA-dependent but JmjC-independent manner, while its JmjC domain mediated removal of H3K9me from established ectopic heterochromatin. Our results suggest that Epe1 not only limits the distribution of heterochromatin but also controls the balance between suppression and retention of heterochromatin-mediated epigenetic diversification.

Indoor Positioning System Based on Chest-Mounted IMU.

Demand for indoor navigation systems has been rapidly increasing with regard to location-based services. As a cost-effective choice, inertial measurement unit (IMU)-based pedestrian dead reckoning (PDR) systems have been developed for years because they do not require external devices to be installed in the environment. In this paper, we propose a PDR system based on a chest-mounted IMU as a novel installation position for body-suit-type systems. Since the IMU is mounted on a part of the upper body, the framework of the zero-velocity update cannot be applied because there are no periodical moments of zero velocity. Therefore, we propose a novel regression model for estimating step lengths only with accelerations to correctly compute step displacement by using the IMU data acquired at the chest. In addition, we integrated the idea of an efficient map-matching algorithm based on particle filtering into our system to improve positioning and heading accuracy. Since our system was designed for 3D navigation, which can estimate position in a multifloor building, we used a barometer to update pedestrian altitude, and the components of our map are designed to explicitly represent building-floor information. With our complete PDR system, we were awarded second place in 10 teams for the IPIN 2018 Competition Track 2, achieving a mean error of 5.2 m after the 800 m walking event.

Reconstruction-Based Change Detection with Image Completion for a Free-Moving Camera.

Reconstruction-based change detection methods are robust for camera motion. The methods learn reconstruction of input images based on background images. Foreground regions are detected based on the magnitude of the difference between an input image and a reconstructed input image. For learning, only background images are used. Therefore, foreground regions have larger differences than background regions. Traditional reconstruction-based methods have two problems. One is over-reconstruction of foreground regions. The other is that decision of change detection depends on magnitudes of differences only. It is difficult to distinguish magnitudes of differences in foreground regions when the foreground regions are completely reconstructed in patch images. We propose the framework of a reconstruction-based change detection method for a free-moving camera using patch images. To avoid over-reconstruction of foreground regions, our method reconstructs a masked central region in a patch image from a region surrounding the central region. Differences in foreground regions are enhanced because foreground regions in patch images are removed by the masking procedure. Change detection is learned from a patch image and a reconstructed image automatically. The decision procedure directly uses patch images rather than the differences between patch images. Our method achieves better accuracy compared to traditional reconstruction-based methods without masking patch images.

Yield Visualization Based on Farm Work Information Measured by Smart Devices.

This paper proposes a new approach to visualizing spatial variation of plant status in a tomato greenhouse based on farm work information operated by laborers. Farm work information consists of a farm laborer's position and action. A farm laborer's position is estimated based on radio wave strength measured by using a smartphone carried by the farm laborer and Bluetooth beacons placed in the greenhouse. A farm laborer's action is recognized based on motion data measured by using smartwatches worn on both wrists of the farm laborer. As experiment, harvesting information operated by one farm laborer in a part of a tomato greenhouse is obtained, and the spatial distribution of yields in the experimental field, called a harvesting map, is visualized. The mean absolute error of the number of harvested tomatoes in each small section of the experimental field is 0.35. An interview with the farm manager shows that the harvesting map is useful for intuitively grasping the states of the greenhouse.

Tethering an N-Glycosylation Sequon-Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex.

Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the Asn residue in the Asn-X-Ser/Thr sequon in proteins, where X is not proline. A sequon was tethered to an archaeal OST enzyme via a disulfide bond. The positions of the cysteine residues in the OST protein and the sequon-containing acceptor peptide were selected by reference to the eubacterial OST structure in a noncovalent complex with an acceptor peptide. We determined the crystal structure of the cross-linked OST-sequon complex. The Ser/Thr-binding pocket recognizes the Thr residue in the sequon, and the catalytic structure termed the "carboxylate dyad" interacted with the Asn residue. Thus, the recognition and the catalytic mechanism of the sequon are conserved between the archaeal and eubacterial OSTs. We found that the tethered peptides in the complex were efficiently glycosylated in the presence of the oligosaccharide donor. The stringent requirements are greatly relaxed in the cross-linked state. The two conserved acidic residues in the catalytic structure were each dispensable, although the double mutation abolished the activity. A Gln residue at the Asn position in the sequon functioned as an acceptor, and the hydroxy group at position +2 was not required. In the standard assay using short free peptides, strong amino acid preferences were observed at the X position, but the preferences, except for Pro, completely disappeared in the cross-linked state. By skipping the initial binding process and stabilizing the complex state, the catalytically competent cross-linked complex offers a unique system for studying the oligosaccharyl transfer reaction.

Adapting Local Features for Face Detection in Thermal Image.

A thermal camera captures the temperature distribution of a scene as a thermal image. In thermal images, facial appearances of different people under different lighting conditions are similar. This is because facial temperature distribution is generally constant and not affected by lighting condition. This similarity in face appearances is advantageous for face detection. To detect faces in thermal images, cascade classifiers with Haar-like features are generally used. However, there are few studies exploring the local features for face detection in thermal images. In this paper, we introduce two approaches relying on local features for face detection in thermal images. First, we create new feature types by extending Multi-Block LBP. We consider a margin around the reference and the generally constant distribution of facial temperature. In this way, we make the features more robust to image noise and more effective for face detection in thermal images. Second, we propose an AdaBoost-based training method to get cascade classifiers with multiple types of local features. These feature types have different advantages. In this way we enhance the description power of local features. We did a hold-out validation experiment and a field experiment. In the hold-out validation experiment, we captured a dataset from 20 participants, comprising 14 males and 6 females. For each participant, we captured 420 images with 10 variations in camera distance, 21 poses, and 2 appearances (participant with/without glasses). We compared the performance of cascade classifiers trained by different sets of the features. The experiment results showed that the proposed approaches effectively improve the performance of face detection in thermal images. In the field experiment, we compared the face detection performance in realistic scenes using thermal and RGB images, and gave discussion based on the results.

Phosphorylation of Swi6/HP1 regulates transcriptional gene silencing at heterochromatin.

Heterochromatin protein 1 (HP1) recruits various effectors to heterochromatin for multiple functions, but its regulation is unclear. In fission yeast, a HP1 homolog Swi6 recruits SHREC, Epe1, and cohesin, which are involved in transcriptional gene silencing (TGS), transcriptional activation, and sister chromatid cohesion, respectively. We found that casein kinase II (CK2) phosphorylated Swi6. Loss of CK2-dependent Swi6 phosphorylation alleviated heterochromatic TGS without affecting heterochromatin structure. This was due to the inhibited recruitment of SHREC to heterochromatin, accompanied by an increase in Epe1. Interestingly, loss of phosphorylation did not affect cohesion. These results indicate that CK2-dependent Swi6 phosphorylation specifically controls TGS in heterochromatin.

Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation.

Oligosaccharyltransferase transfers an oligosaccharide chain to the asparagine residues in proteins. The archaeal and eubacterial oligosaccharyltransferases are single subunit membrane enzymes, referred to as "AglB" (archaeal glycosylation B) and "PglB" (protein glycosylation B), respectively. Only one crystal structure of a full-length PglB has been solved. Here we report the crystal structures of the full-length AglB from a hyperthermophilic archaeon, Archaeoglobus fulgidus. The AglB and PglB proteins share the common overall topology of the 13 transmembrane helices, and a characteristic long plastic loop in the transmembrane region. This is the structural basis for the formation of the catalytic center, consisting of conserved acidic residues coordinating a divalent metal ion. In one crystal form, a sulfate ion was bound next to the metal ion. This structure appears to represent a dolichol-phosphate binding state, and suggests the release mechanism for the glycosylated product. The structure in the other crystal form corresponds to the resting state conformation with the well-ordered plastic loop in the transmembrane region. The overall structural similarity between the distantly related AglB and PglB proteins strongly indicates the conserved catalytic mechanism in the eukaryotic counterpart, the STT3 (stauroporine and temperature sensitivity 3) protein. The detailed structural comparison provided the dynamic view of the N-glycosylation reaction, involving the conversion between the structured and unstructured states of the plastic loop in the transmembrane region and the formation and collapse of the Ser/Thr-binding pocket in the C-terminal globular domain.

Minimally invasive elective gastrectomy after preoperative chemotherapy in a patient with frailty who presented with locally far advanced-stage gastric cancer: a case report.

BACKGROUND: Herein, we report a case of gastric antrum cancer with multiple invasions to other organs that was completely cured with laparoscopic distal gastrectomy after preoperative chemotherapy in a patient with poor general condition. CASE PRESENTATION: An 80-year-old male patient was diagnosed with anemia during follow-up for cerebral lacunar infarction at another hospital. He was diagnosed with advanced-stage gastric antrum cancer and was referred to our hospital. On esophagogastroduodenoscopy, type 2 advanced-stage gastric cancer was detected at the greater curvature of the antrum, and the biopsy results revealed tubular adenocarcinoma. Contrast-enhanced computed tomography scan revealed multiple invasions to other organs, thick gastric wall with contrast effect, and superior mesenteric vein tumor thrombus. However, there was no evidence of distant metastasis on positron emission tomography/computed tomography scan. The clinical diagnosis was stage IVA gastric cancer. Pancreatoduodenectomy with portal vein resection could be important at this point. However, preoperative chemotherapy with S-1 and oxaliplatin was administered instead of performing extended surgery because the patient had poor general condition (performance status score of 3). The patient received three cycles of preoperative chemotherapy at the hospital along with rehabilitation and nutritional management with oral nutritional supplements. After treatment, the performance status score of the patient improved from 3 to 1. Furthermore, in terms of clinical therapeutic effect, the patient achieved partial response. Hence, laparoscopic distal gastrectomy with D2 lymph node dissection and partial transverse colectomy was performed. After surgery, the patient was admitted for oral intake on postoperative day 6 and was discharged on postoperative day 21. Based on the histopathological examination, gastric cancer had disappeared, and there were no evident malignant findings. Therefore, gastric cancer was classified as grade 3 according to the histological treatment efficacy criteria. The patient did not present with recurrence at 2 years after surgery. CONCLUSIONS: By actively administering preoperative chemotherapy, minimally invasive radical surgery with maximum preservation of the surrounding organs can be performed for locally far advanced-stage gastric cancer in older patients with poor general condition.

Crystal structure of the C-terminal globular domain of the third paralog of the Archaeoglobus fulgidus oligosaccharyltransferases.

BACKGROUND: Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the asparagine residue in the N-glycosylation sequons. The catalytic subunits of the OST enzyme are STT3 in eukaryotes, AglB in archaea and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three paralogous AglB proteins. We previously solved the crystal structures of the C-terminal globular domains of two paralogs, AglB-Short 1 and AglB-Short 2. RESULTS: We determined the crystal structure of the C-terminal globular domain of the third AglB paralog, AglB-Long, at 1.9 Å resolutions. The crystallization of the fusion protein with maltose binding protein (MBP) afforded high quality protein crystals. Two MBP-AglB-L molecules formed a swapped dimer in the crystal. Since the fusion protein behaved as a monomer upon gel filtration, we reconstituted the monomer structure from the swapped dimer by exchanging the swapped segments. The C-terminal domain of A. fulgidus AglB-L includes a structural unit common to AglB-S1 and AglB-S2. This structural unit contains the evolutionally conserved WWDYG and DK motifs. The present structure revealed that A. fulgidus AglB-L contained a variant type of the DK motif with a short insertion, and confirmed that the second signature residue, Lys, of the DK motif participates in the formation of a pocket that binds to the serine and threonine residues at the +2 position of the N-glycosylation sequon. CONCLUSIONS: The structure of A. fulgidus AglB-L, together with the two previously solved structures of AglB-S1 and AglB-S2, provides a complete overview of the three AglB paralogs encoded in the A. fulgidus genome. All three AglBs contain a variant type of the DK motif. This finding supports a previously proposed rule: The STT3/AglB/PglB paralogs in one organism always contain the same type of Ser/Thr-binding pocket. The present structure will be useful as a search model for molecular replacement in the structural determination of the full-length A. fulgidus AglB-L.

The modified Glasgow prognostic score is a reliable predictor of oncological outcomes in patients with rectal cancer undergoing neoadjuvant chemoradiotherapy.

There has been no reliable marker for predicting oncological outcomes in patients with locally advanced rectal cancer (LARC) undergoing neoadjuvant chemoradiotherapy (NACRT). We retrospectively analyzed 73 patients with LARC who underwent curative surgery after NACRT. The modified Glasgow prognostic score (mGPS) was assessed after NACRT, and clinical outcomes were compared between the high (mGPS = 1 or 2; n = 23) and low (mGPS = 0; n = 50) groups. Body mass index was significantly higher in the low mGPS group. The 5-year disease-free survival (DFS) rate was significantly worse in the high mGPS group than that in the low mGPS group (36.7% vs. 76.6%, p = 0.002). Univariate and multivariate analyses of DFS revealed that mGPS was the most significant predictor (p < 0.001). mGPS appears to be a reliable predictor of oncological outcomes in patients with LARC undergoing NACRT.

Retrotransposon addiction promotes centromere function via epigenetically activated small RNAs.

Retrotransposons have invaded eukaryotic centromeres in cycles of repeat expansion and purging, but the function of centromeric retrotransposons, if any, has remained unclear. In Arabidopsis, centromeric ATHILA retrotransposons give rise to epigenetically activated short interfering RNAs (easiRNAs) in mutants in DECREASE IN DNA METHYLATION1 (DDM1), which promote histone H3 lysine-9 di-methylation (H3K9me2). Here, we show that mutants which lose both DDM1 and RNA dependent RNA polymerase (RdRP) have pleiotropic developmental defects and mis-segregation of chromosome 5 during mitosis. Fertility defects are epigenetically inherited with the centromeric region of chromosome 5, and can be rescued by directing artificial small RNAs to a single family of ATHILA5 retrotransposons specifically embedded within this centromeric region. easiRNAs and H3K9me2 promote pericentromeric condensation, chromosome cohesion and proper chromosome segregation in mitosis. Insertion of ATHILA silences transcription, while simultaneously making centromere function dependent on retrotransposon small RNAs, promoting the selfish survival and spread of centromeric retrotransposons. Parallels are made with the fission yeast S. pombe, where chromosome segregation depends on RNAi, and with humans, where chromosome segregation depends on both RNAi and HELLSDDM1.

Reevaluation of serum p53 antibody as a tumor marker in colorectal cancer patients.

PURPOSE: We reevaluated the serum p53 antibody (S-p53Ab) ELISA kit, which was approved as a tumor marker of colon cancer in the Japanese Health Insurance System in 2007. METHODS: S-p53Ab was measured as a tumor marker in 154 colorectal cancer patients, and the results were categorized by clinical and pathological variables. We then compared the positive frequency of S-p53Ab, carcinoembryonic antigen (CEA), and carbohydrate 19-9 (CA19-9). RESULTS: S-p53Ab was positive in 33.1% of the colorectal cancer patients. The positive rate was significantly higher in patients with lymph nodes metastasis (P = 0.025) and lymphatic invasion (P = 0.023). In patients with stage I colorectal cancer, the positive rate of S-p53Ab (23.7%) was significantly higher than that of CEA (5.3%) or CA19-9 (7.9%). CONCLUSION: The approved kit for S-p53Ab testing was found to be an effective tumor marker of colorectal cancer. The positive rate of S-p53Ab was significantly higher in patients with cancer involvement of the lymphoid tissues. The positive rate of S-p53Ab was higher than that of CEA and CA19-9 in patients with stage I colorectal cancer, suggesting that the S-p53Ab is a useful tumor marker for patients with early-stage disease.

Effects of targeting signal mutations in a mitochondrial presequence on the spatial distribution of the conformational ensemble in the binding site of Tom20.

The 20-kDa TOM (translocase of outer mitochondrial membrane) subunit, Tom20, is the first receptor of the protein import pathway into mitochondria. Tom20 recognizes the mitochondrial targeting signal embedded in the presequences attached to mature mitochondrial proteins, as an N-terminal extension. Consequently, ~1,000 different mitochondrial proteins are sorted into the mitochondrial matrix, and distinguished from non-mitochondrial proteins. We previously reported the MPRIDE (multiple partial recognitions in dynamic equilibrium) mechanism to explain the structural basis of the promiscuous recognition of presequences by Tom20. A subset of the targeting signal features is recognized in each pose of the presequence in the binding state, and all of the features are collectively recognized in the dynamic equilibrium between the poses. Here, we changed the volumes of the hydrophobic side chains in the targeting signal, while maintaining the binding affinity. We tethered the mutated presequences to the binding site of Tom20 and placed them in the crystal contact-free space (CCFS) created in the crystal lattice. The spatial distributions of the mutated presequences were visualized as smeared electron densities in the low-pass filtered difference maps obtained by X-ray crystallography. The mutated presequence ensembles shifted their positions in the binding state to accommodate the larger side chains, thus providing positive evidence supporting the use of the MPRIDE mechanism in the promiscuous recognition by Tom20.

Ca2+ -induced structural changes and intramolecular interactions in N-terminal region of diacylglycerol kinase alpha.

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca2+ -triggered activation of DGKα, for which the N-terminal domains likely play a critical role, remains unclear. We have recently shown that Ca2+ binding to DGKα-EF induces conformational changes from a protease-susceptible "open" conformation in the apo state to a well-folded one in its holo state. Here, we further studied the structural properties of DGKα N-terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N-terminal RVH domain is a novel Ca2+ -binding domain, but the Ca2+ -induced conformational changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten-globule like structure in the apo state. Further analyses using SEC-SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydrophobic interactions in the absence of Ca2+ , and this interaction is modified in the presence of Ca2+ . Taken together, these results present novel insights into the structural rearrangement of DGKα N-terminal domains upon binding to Ca2+ , which is essential for the activation of the enzyme.