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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer–RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.

Few data exist regarding steroid withdrawal in ABO-incompatible (ABO-i) kidney transplantation (KT). Here, we report a case of steroid withdrawal after ABO-i KT. A 46-year-old man diagnosed with Henoch-Schonlein purpura received ABO-i KT from his 42-year-old sister. The recipient and donor blood types were O and AB, respectively. His preoperative ABO antibody titers were anti-A of 1:16 and anti-B of 1:8 in isoaggluti nin test. HLA mismatch was 0 and he received a single 325 mg/m2 dose of intravenous (IV) rituximab 4 weeks before KT. Three sessions of plasma exchange were undertaken before KT and low-dose IV immunoglobulin of 0.1 g/kg was administered after plasma exchange. On the day of the operation, ABO antibody titer decreased to anti-A of 1:4 and anti-B of 1:2. Renal function remained stable after KT. The patient wished to stop ste roid treatment despite the risk of rejection after withdrawal. Steroid tapering was initi ated at 20 months and accomplished at 26 months after KT. At that time, serum creati nine level was 1.13 mg/dL, and anti-A and anti-B titers were 1:8 and 1:2, respectively. No issues were observed after steroid withdrawal. At 48 months after KT, serum creatinine level was 1.21 mg/dL, and anti-A and anti-B antibody titers were 1:32 and 1:2, respec tively. Steroid withdrawal in ABO-i KT might be considered in immunologically low-risk patients.

Rylene dyes exhibit superior photophysical properties including outstanding chemical and photochemical stabilities as well as high fluorescence quantum yields (FQYs). However, the rylene chromophores have some limitations, such as high hydrophobicity and low solubility in aqueous solution, as a fluorescent tag for biological system. Herein, we present a general method to load the rylene dyes into triblock copolymer nanoparticles (NPs) and stabilize the core. The rylene dyes in confined hydrophobic core fairly preserve their FQYs in aqueous environment. Lumogen probe nanoparticles (PNPs) were prepared with four different commercial rylenes (Lumogen Violet, Yellow, Orange and Red) in the spectral region of visible and infrared (400–750 nm) and their photophysical properties were characterized. They exhibited enhanced photostability compared to commercial fluorescent dyes. Also the internalization of Lumogen PNPs in HeLa cell was confirmed by cell uptake test. These results indicate the Lumogen PNPs can be widely applicable in fluorescence detection and imaging such as flow cytometry and fluorescence microscopy.

Esophageal cancer is one of the lethal malignant tumors because it is often diagnosed in the advanced stage with lymph node or distant metastasis. The recurrence rate of esophageal cancer is known to be about 40% to 50% even in the patients who underwent curative esophageal resection. Most frequent sites of metastases are known to be the liver. Furthermore, other distant metastases also could be developed in lung, bone, brain, kidney, adrenal, abdominal cavity, and skin. However, ampulla of Vater (AoV) metastasis rarely occurs from esophageal cancer. Therefore, we report herein a case of AoV metastasis from squamous cell carcinoma of the esophagus.

Circulating tumor DNA (ctDNA) has emerged as a candidate biomarker for cancer screening. However, studies on the usefulness of ctDNA for postoperative recurrence monitoring are limited. The present study monitored ctDNA in postoperative blood by employing cancer-specific rearrangements. Personalized cancer-specific rearrangements in 25 gastric cancers were analyzed by whole-genome sequencing (WGS) and were employed for ctDNA monitoring with blood up to 12 months after surgery. Personalized cancer-specific rearrangements were identified in 19 samples. The median lead time, which is the median duration between a positive ctDNA detection and recurrence, was 4.05 months. The presence of postoperative ctDNA prior to clinical recurrence was significantly correlated with cancer recurrence within 12 months of surgery (P = 0.029); in contrast, no correlation was found between cancer recurrence and the presence of preoperative ctDNA, suggesting the clinical usefulness of postoperative ctDNA monitoring for cancer recurrence in gastric cancer patients. However, the clinical application of ctDNA can be limited by the presence of ctDNA non-shedders (42.1%, 8/19) and by inconsistent postoperative ctDNA positivity., Cancer: Tell-tale signs of recurrence Fragments of tumor DNA, or circulating tumor DNA (ctDNA), in blood can help predict stomach cancer recurrence within 12 months of surgery. Kyeong-Man Hong at the National Cancer Center, in Goyang-si, South Korea, and colleagues, carried out whole genome sequencing of stomach tumor samples from 25 patients to identify personalized cancer-specific rearranged DNA sequences. When they used this information to monitor ctDNA in blood samples obtained after surgical removal of the tumor, they found a significant correlation between the presence of ctDNA and cancer recurrence. In most cases, ctDNA was detected around four months prior to clinical recurrence, highlighting the potential usefulness of ctDNA monitoring. The lack of correlation between ctDNA levels and tumor size suggests that further research into the factors determining ctDNA levels is needed.

Polymeric micelles are versatile nanocarriers to carry hydrophobic moieties within the core in aqueous media. We report engineered polymeric micelles encapsulating highly photostable organic near-i...

Pluronic® triblock copolymers, which spontaneously form nano-sized aggregates in water, have been considered as promising carriers for bioimaging and drug delivery. However, their use for applications in pharmacology and diagnostics is hindered by instability of polymer aggregates which are easily dissociated into unimers. Here we report a general method for stabilizing Pluronic® F127 micelles via semi-interpenetrating network (sIPN). The formation of sIPN within a core stabilizes the micelle upon temperature and concentration changes. We determined optimized methods for the preparation of F127 sIPN in the regime of chemical components. Importantly, F127 sIPNs are able to load various organic fluorescence probes, covering a panchromatic range of photoluminescence (350–850 nm; entire UV–Vis-NIR), without compromising their photophysical properties. Moreover, efficient cellular uptake of the fluorescence probes loaded sIPN is observed in human and mouse cells. After intravenous injection into mice, an infrared dye-loaded sIPNs are incorporated in multiple organs and have longer lifetime than a commercially available imaging probe. With the improvement of stability, Pluronic micelles with sIPN can be a powerful tool for building functional nanoprobes and in vivo fluorescence imaging for cancer cells and live animals.

Background Although surgical resection with chemotherapy is considered effective for patients with advanced gastric cancer, it remains the third leading cause of cancer-related death in South Korea. Several studies have reported that mesothelial markers including mesothelin, calretinin, and Wilms tumor protein 1 (WT1) were positive in variable carcinomas, associated with prognosis, and were evaluated as potential markers for targeted therapy. The aim of this study was to assess the immunohistochemical expression of mesothelial markers (mesothelin, calretinin, and WT1) in gastric adenocarcinoma and their relations to clinocopathological features and prognosis. Methods We evaluated calretinin, WT1, and mesothelin expression by immunohistochemical staining in 117 gastric adenocarcinomas. Results Mesothelin was positively stained in 30 cases (25.6%). Mesothelin expression was related to increased depth of invasion (p = .002), lymph node metastasis (p = .013), and presence of lymphovascular (p = .015) and perineural invasion (p = .004). Patients with mesothelin expression had significantly worse disease-free survival rate compared with that of nonmesothelin expression group (p = .024). Univariate analysis showed that mesothelin expression is related to short-term survival. None of the 117 gastric adenocarcinomas stained for calretinin or WT1. Conclusions Mesothelin expression was associated with poor prognosis. Our results suggest that mesothelin-targeted therapy should be considered as an important therapeutic alternative for gastric adenocarcinoma patients with mesothelin expression.

Summary Mitochondrial sulfide quinone oxidoreductase (SQR) catalyzes the oxidation of H2S to glutathione persulfide with concomitant reduction of CoQ10. We report herein that the promiscuous activity of human SQR supported the conversion of CoA to CoA-SSH (CoA-persulfide), a potent inhibitor of butyryl-CoA dehydrogenase, and revealed a molecular link between sulfide and butyrate metabolism, which are known to interact. Three different CoQ1-bound crystal structures furnished insights into how diverse substrates access human SQR, and provided snapshots of the reaction coordinate. Unexpectedly, the active site cysteines in SQR are configured in a bridging trisulfide at the start and end of the catalytic cycle, and the presence of sulfane sulfur was confirmed biochemically. Importantly, our study leads to a mechanistic proposal for human SQR in which sulfide addition to the trisulfide cofactor eliminates 201Cys-SSH, forming an intense charge-transfer complex with flavin adenine dinucleotide, and 379Cys-SSH, which transfers sulfur to an external acceptor.

Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

SUMMARYMixed Lineage Leukemia (MLL) family histone methyltransferases are the key enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanism by which the MLL complexes recognize histone H3K4 within the nucleosome core particle (NCP) remains unclear. Here, we report the single-particle cryo-electron microscopy (cryo-EM) structure of the human MLL1 core complex bound to the NCP. The MLL1 core complex anchors on the NCP through RbBP5 and ASH2L, which interacts extensively with nucleosomal DNA as well as the surface close to histone H4 N-terminal tail. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, allowing symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

Hydrogen sulfide (H(2)S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H(2)S synthesis and is a potential target for modulating H(2)S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H(2)S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H(2)S synthesis from cysteine by human CSE with K(i) values of 50 ± 3 and 180 ± 15 μm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine β-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H(2)S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80–90%, as monitored by the transfer of radiolabel from [(35)S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H(2)S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H(2)S biogenesis inhibitors.

Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity., MLL family histone methyltransferases deposit histone H3 Lys4 mono-/di-/tri-methylation and regulate gene expression in mammals. Here the authors report the single-particle cryo-EM structure of the NCP-bound human MLL1 core complex, shedding light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

SummarySoluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions1-3. The maximum catalytic activity of hydroxylase (MMOH) is achieved via interplay of its regulatory protein (MMOB) and reductase4-6. An additional auxiliary protein, MMOD, is believed to function as an inhibitor of the catalytic activity of MMOH; however, the mechanism of its action remains unknown7,8. Herein, we report the crystal structure of MMOH–MMOD complex fromMethylosinus sporiumstrain 5 (2.6 Å), which illustrates that two molecules of MMOD associate symmetrically with the canyon region of MMOH in a manner similar to MMOB, indicating that MMOD competes with MMOB for MMOH recognition. Further, MMOD binding disrupts the geometry of the di-iron centre and opens the substrate access channel. Notably, the electron density of 1,6-hexanediol at the substrate access channel mimics products of sMMO in hydrocarbon oxidation. The crystal structure of MMOH–MMOD unravels the inhibitory mechanism by which MMOD suppresses the MMOH catalytic activity, and reveals how hydrocarbon substrates/products access to the di-iron centre.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

CENP-A is a centromere-specific histone H3 variant that is required for kinetochore assembly and accurate chromosome segregation. For it to function properly, CENP-A must be specifically localized to centromeres. In fission yeast, Scm3sp and the Mis18 complex, composed of Mis16, Eic1, and Mis18, function as a CENP-A(Cnp1)-specific chaperone and a recruiting factor, respectively, and together ensure accurate delivery of CENP-A(Cnp1) to centromeres. Although how Scm3sp specifically recognizes CENP-A(Cnp1) has been revealed recently, the recruiting mechanism of CENP-A(Cnp1) via the Mis18 complex remains unknown. In this study, we have determined crystal structures of Schizosaccharomyces japonicus Mis16 alone and in complex with the helix 1 of histone H4 (H4α1). Crystal structures followed by mutant analysis and affinity pull-downs have revealed that Mis16 recognizes both H4α1 and Scm3sp independently within the CENP-A(Cnp1)/H4:Scm3sp complex. This observation suggests that Mis16 gains CENP-A(Cnp1) specificity by recognizing both Scm3sp and histone H4. Our studies provide insights into the molecular mechanisms underlying specific recruitment of CENP-A(Cnp1)/H4:Scm3sp into centromeres.

SummaryIn proteins, functional divergence involves mutations that modify structure and dynamics. Here we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the β barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.

Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kluyveromyces lactis Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.

Multiple wasp stings can induce multiple organ dysfunction by toxic reactions. However, acute pancreatitis is a rare manifestation in wasp sting injury. A 74-year-old woman visited the emergency department by anaphylactic shock because of multiple wasp stings. Acute kidney injury, rhabdomyolysis, hepatotoxicity, and coagulopathy were developed next day. Serum amylase and lipase were elevated and an abdominal computed tomography revealed an acute pancreatitis. Urine output was recovered after 16 days of oliguria (below 500 ml/day). Her kidney, liver, and pancreas injury gradually improved after sessions of renal replacement therapy.

Background Recently, we reported cytoskeleton-associated protein2 (CKAP2) as a possible new prognostic breast cancer marker. However, it has not yet been applied in clinic. Therefore, clinical significance of CKAP2 was evaluated in comparison with that of Ki-67 in a cohort of breast cancer patients, and the expression difference was analyzed in cell cycle-arrested cancer and fibroblast cells. Methods A total of 579 early breast cancer patients who underwent surgery at the National Cancer Center Hospital in Korea between 2001 and 2005 were accrued. CKAP2-positive cell count (CPCC) and Ki-67 labeling index (Ki-67LI) were evaluated by immunohistochemcal staining. The immunocytochemical staining patterns of CKAP2 and Ki-67 were analyzed in HeLa and human fibroblast cells after synchronization by double thymidine block. Results Although there was a significant correlation (R = 0.754, P < 0.001) between CPCC and Ki-67LI, only CPCC was correlated with DFS in overall population (HR, 2.029; 95% CI, 1.012–4.068; P = 0.046) and HER2-negative luminal subgroup (HR, 3.984; 95% CI, 1.350–11.762; P = 0.012) by multivariate analysis. In immunocytochemical staining, more than 50% of serum-starved or non-mitotic cell phase HeLa cells were positive for Ki-67, in comparison to the low CKAP2-positivity, which might explain the prognostic difference between CPCC and Ki-67LI. Conclusions The current study showed that CPCC but not Ki-67LI is an independent prognostic indicator in early breast cancer, more specifically in HER2-negative luminal breast cancer. The difference between two markers may be related to the lower background expression of CKAP2 in cancer cells.

Malakoplakia, meaning soft (malako) plaque (plakia), is a rare benign inflammatory lesion. The disease was initially described in the bladder by Michaelis and Gutmann [1] in 1902 and named by von Hansemann [2] in 1903. Since its initial description, many cases have been described in numerous anatomic locations including the genitourinary tract, gastrointestinal tract, pancreas, liver, oropharynx, retroperitoneum, thyroid gland, lymph nodes, lung, bone/joint, brain, skin, and other tissues [3-5]. To our knowledge, this is the first case of malacoplakia affecting the umbilical cord.

In green-to-red photoconvertible fluorescent proteins, a three-ring chromophore is generated by the light-activated incorporation of a histidine residue into the conjugated π-system. We have determined the pH-rate profile and high- and low-pH X-ray structures of a least evolved ancestor (LEA) protein constructed in the laboratory based on statistical sequence analysis. LEA incorporates the minimal number of substitutions necessary and sufficient for facile color conversion and exhibits a maximal photoconversion quantum yield of 0.0015 at pH 6.1. The rate measurements provide a bell-shaped curve, indicating that the reaction is controlled by the two apparent pKa values, 4.5 ± 0.2 and 7.5 ± 0.2, flanking the chromophore pKa of 6.3 ± 0.1. These data demonstrate that the photoconversion rate of LEA is not proportional to the A-form of the GFP-like chromophore, as previously reported for Kaede-type proteins. We propose that the observed proton dissociation constants arise from the internal quadrupolar charge network consisting of Glu222, His203, Glu148, and Arg69. Increased active site flexibility may facilitate twisting of the chromophore upon photoexcitation, thereby disrupting the charge network and activating the Glu222 carboxylate for the abstraction of a proton from a carbon acid. Subsequently, the proton may be delivered to the Phe64 carbonyl by a hydrogen-bonded network involving Gln42 or by means of His65 side chain rotations promoted by protein breathing motions. A structural comparison of LEA with the nonphotoconvertible LEA-Q42A variant supports a role for Gln42 either in catalysis or in the coplanar preorganization of the green chromophore with the His65 imidazole ring.

Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.

Granular cell astrocytoma is a rare infiltrative malignant glioma with prominent granular cell change. Granular cell astrocytomas are biologically aggressive compared with conventional infiltrating astrocytomas of similar grades, but their genetic alterations are poorly known. We report a case of granular cell glioblastoma and its genetic and molecular features. Histologically, the tumor not only showed features typical of granular cell astrocytoma but also demonstrated frequent mitoses, pseudopalisading necrosis, and vascular endothelial hyperplasia, compatible with glioblastoma. Array-based comparative genomic hybridization and focused molecular genetic analyses demonstrated gain of chromosome 7; losses of chromosome 1p, 8p, 9p, 10, 13q, and 22q; amplification of epidermal growth factor receptor; and homozygous deletion of CDKN2A as well as MGMT promoter methylation. However, neither isocitrate dehydrogenase 1 mutation nor codeletion of 1p/19q was found. Our results indicate that granular cell glioblastomas, despite having its peculiar granular cell changes, share common molecular genetic features with conventional glioblastoma, especially the classical subtype.

In this study, the instabilities of electrospinning were observed and evaluated by measuring the drop area, the current, and the electrical force using PVAc and PVA. Those values were measured simultaneously during electrospinning and the instabilities were analyzed as a function of time. The electrospinning was carried out by changing the voltages and the feed rates. The results suggest that different electrospinning conditions such as voltage, polymer, and flow rate caused different behaviors of instabilities, current and electric force, however, all the used methods were effective for studying the instabilities in electrospinning.

A 53-year-old man presented with right flank pain for 6 days. Computerized tomography revealed a 3 cm long segment of ureteral narrowing with wall thickening and hydronephrosis, suspicious for ureteral cancer. Under the clinical diagnosis of ureteral carcinoma a right nephroureterectomy was performed. The wall of the distal ureter, 2.5 cm from the bladder cuff, had a luminal-narrowing, firm mass-forming lesion with abrupt transition from the adjacent ureter. Histologically, the resected ureteral mass showed transmural fibrosing, chronic inflammation with numerous plasma cells, epithelioid granulomas, and obliterative phlebitis. Histological findings were consistent with idiopathic segmental ureteritis (ISU) with differential diagnoses of IgG4-related sclerosing disease, including lymphoplasmacytic inflammatory pseudotumor (IPT) and idiopathic retroperitoneal fibrosis. IgG4 immunostaining in this case was barely positive, excluding the possibility of IgG4-related IPT. Although the majority of luminal obliterated segmental lesions of the ureter are neoplastic in nature, non-neoplastic inflammatory processes as seen in this case may occur in the ureter, causing diagnostic confusion with true neoplasms. Herein we report a rare case of ISU that was clinically misdiagnosed as malignancy preoperatively. ISU of the current case may be an IgG4-unrelated subtype of IPT.

Selective IgA deficiency is the most common primary immunoglobulin deficiency. The clinical manifestations of selective IgA deficiency, including gastrointestinal (GI) complications, are rare and typically milder than those seen with common variable immunodeficiency or X-linked agammaglobulinemia. We present a rare case of selective IgA deficiency that shows a number of interesting histological features in the GI tract, including diffuse nodular lymphoid hyperplasia involving the entire small and large intestine, celiac disease-like and collagenous sprue-like changes in the small intestine, as well as lymphocytic colitis pattern. However, this patient had no particular GI symptoms suggestive of celiac sprue or microscopic colitis. These findings suggest that the GI tract in patients with selective IgA deficiency can show peculiar histologic changes that mimic celiac disease, collagenous sprue, or lymphocytic colitis, which may be a pattern of injury related to infection or immunoglobulin immunodeficiency-associated autoimmune phenomena.

Clear cell sarcoma (CCS) is a distinctive soft tissue sarcoma that shows melanocytic differentiation. Primary gastrointestinal (GI) CCSs have been rarely reported, but to our knowledge, no association between GI CCSs and immunoglobulin G4 (IgG4)-related sclerosing disease has been described in the literature. We experienced 2 cases of CCS that arose in the small intestine and metastasized to the liver. Histologic features and immunophenotype were typical of CCS. One of them showed a unique peritumoral sclerosing inflammatory reaction, which was highly reminiscent of IgG4-related sclerosing inflammatory disease. Dense lymphoplasmacytic infiltration with extensive sclerosis and obliterative phlebitis was observed in the immediate vicinity of the primary and metastatic tumors, but not in the distant areas from the tumor. The average number of IgG4-positive plasma cells was more than 50 per high-power field. We report 2 cases of primary GI CCS with one case showing a unique peritumoral IgG4-related lymphoplasmacytic sclerosing inflammation.

Background and aim: Although morphological criteria are important for diagnosis of cervical intraepithelial neoplasia, it is not possible to differentiate those that will regress from those that will progress into invasive cancer. We evaluate the usefulness of p16, cyclin D1, and human telomerase reverse transcriptase (hTERT) as diagnostic and prognostic biomarkers in cervical lesions. Methods: We studied 12 cases of benign lesion, 15 cases of low-grade squamous intraepithelial lesion, 46 cases of high-grade squamous intraepithelial lesion, and 13 cases of squamous cell carcinoma by immunohistochemistry. Results: The p16 expression was significantly correlated with high risk-human papillomavirus (HR-HPV) status, lesion grade, and decreased cyclin D1 expression. The decreased cyclin D1 expression was significantly correlated with HR-HPV status, lesion grade, and p16 expression. The positive predictive values for high grade lesion of p16 expression, HR-HPV status, and decreased cyclin D1 expression were 93.8%, 81.3%, and 51.2%, respectively. The distribution pattern of hTERT positive cells was correlated with morphologically dysplastic cells. Conclusions: A good correlation was observed between cervical lesion grade and p16 expression, decreased cyclin D1 expression, and HR-HPV status. p16 expression could be used as a diagnostic and prognostic biomarker for risk of developing cervical cancer in women infected with HPV.

The aims of this study were to evaluate the clinicopathologic features of Helicobacter heilmannii-associated gastritis and to compare H. heilmannii-associated gastritis with H. pylori-associated gastritis. We reviewed 5,985 consecutive gastric biopsy specimens. All cases of chronic gastritis with Helicobacter infection were evaluated with the Updated Sydney System, and the grades of all gastritis variables were compared between H. heilmannii-associated gastritis and H. pylori-associated gastritis groups. There were 10 cases of H. heilmannii-associated gastritis (0.17%) and 3,285 cases of H. pylori-associated gastritis (54.9%). The organisms were superficially located within the mucous layer without adhesion to epithelial cells. Interestingly, in one case many intracytoplasmic H. heilmannii organisms were observed in parietal cells with cell damage. A case of low-grade mucosa-associated lymphoid tissue (MALT) lymphoma concomitant with H. heilmannii infection was detected. Compared to H. pylori-associated gastritis, H. heilmannii-associated gastritis showed less severe neutrophilic activity (p

Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix–turn–helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation., Sestrins are conserved stress-inducible metabolic regulators implicated in the prevention of agerelated diseases. Here, Kim et al. report the crystal structure of human Sestrin2 and propose a molecular mechanism for how Sestrin2 functions to prevent ROS accumulation and inhibit mTORC1 activity.

OBJECT In this study the authors sought to evaluate clinical outcomes after using stereotactic radiosurgery (SRS) to treat benign and malignant spinal neurogenic tumors. METHODS The authors reviewed a total of 66 procedures of spinal SRS performed between 2001 and 2013 for 110 tumors in 58 patients with spinal neurogenic tumors, which included schwannomas, neurofibromas, and malignant peripheral nerve sheath tumors (MPNSTs). The clinical and radiological findings were evaluated in patients with benign neurogenic tumors. For the 4 patients with MPNSTs, the authors reported overall survival and results of additional immunohistochemical staining to predict the survival difference among the patients. RESULTS Of the 92 benign neurogenic tumors, 65 tumors that were serially followed up using MRI after SRS showed significant change in mean tumor volume, from a mean of 12.0 ± 2.6 cm3 pre-SRS to 10.8 ± 2.5 cm3 post-SRS (p = 0.027), over an average of 44 months. The local control rate of benign neurogenic tumors was 95.4%. The 34 patients who presented with clinical symptoms of pain showed a significant symptomatic improvement. The initial mean visual analog scale (VAS) score was 6.0 and decreased dramatically to 1.0 after SRS during an average follow-up period of 10.9 months (median of 8.1 months). Although the proportions of transient swelling and loss of intramural enhancement were significantly different among the groups, there was no statistically significant correlation between those 2 factors and local tumor control (p = 0.253 and 0.067, respectively; Fisher’s exact text). Cross-table analysis also indicated that there was no statistically significant relationship between groups with loss of intramural enhancement and transient swelling. The median survival of neurofibromatosis Type 1 (NF1)-related and sporadic MPNSTs was 1.13 and 5.8 years, respectively. Immunohistochemical results showed that S100 was expressed in a sporadic MPNST or neurofibroma, whereas topoisomerase-IIa was expressed in NF1-related MPNSTs. CONCLUSIONS SRS is an effective treatment modality for benign neurogenic tumors, while MPNSTs showed heterogeneity in their responses to SRS.

Background: The epithelial cell adhesion molecule (Ep-CAM) is widely associated with human carcinomas. However, the expression and distribution of Ep-CAM in gastric premalignant and malignant lesions are not well known. Method: We examined the expression of Ep-CAM in 99 cases of gastric adenocarcinoma and associated uninvolved gastric mucosa, 39 cases of gastric biopsy specimens with chronic gastritis (CG) with or without intestinal metaplasia (IM) (25 cases) and gastric epithelial dysplasia (GED) (14 cases) by immunohistochemical staining. In gastric adenocarcinoma, we correlated the results with established prognostic factors, and in IM and GED, with Hepatocyte paraffin 1 (Hep Par 1) expression, introduced as a marker of IM. Results: Ep-CAM overexpression was noted in 0% of normal epithelia, 93.9% of IM, 42.9% of GED and 34.3% of adenocarcinoma. The average immunostaining score of normal epithelia, IM, GED and gastric adenocarcinoma was 0.14 (± 0.26), 7.18 (± 1.93), 5.67 (± 2.29) and 4.09 (± 1.89), respectively. There were significant differences among the groups. Ep-CAM overexpression in adenocarcinoma correlated with Lauren classification and histologic grade, but not with tumor stage, lymph node metastasis and p53 expression. Both Ep-CAM and Hep Par 1 expressions showed excellent correlations with IM (P

A 41-year-old male presented with an ill-demarcated mass involving the left fronto-temporal lobe and the basal ganglia. Light microscopically the tumor was diagnosed as an anaplastic oligoastrocytoma. Electron microscopy revealed several cytoplasmic crystalline inclusions in tumor cells of oligodendroglial lineage. No crystalline inclusions were membrane bound. The morphologic features with unique ultrastructural findings are presented.

In normal breast, Bcl2 is expressed in the non-pregnant and non-involuting mammary epithelium. The exact mechanism and the effect of the down regulation of the Bcl2 expression on breast cancer cells are not clearly defined. We compared down regulation as well as the persistent expression of Bcl2 with ER, PR, p53, and c-erb-B2 overexpression and clinicopathologic variables, and tumor stage in 11 cases of ductal carcinomas in situ (DCIS) and 44 cases of invasive ductal carcinomas (IDC) of Korean women by immunohistochemical studies. Bcl2 down regulation was found in 39% of IDC and in 18% of DCIS cases. In IDC, while persistent Bcl2 expression was displayed in 95% and 78.9% of ER and PR immunoreactive ones and 71.9 % of c-erb-B2 immnonegative ones. Seventeen cases of Bcl2 down regulated IDC had a significant correlation with ER negativity (94.1%), PR negativity, (76.5%), and high nuclear (61.1% is grade III) and histological grade (76% is grade III). However, in DCIS, no significant correlation between the Bcl2 expression and various parameters were obtained, probably due to small sample size. In conclusion, the Bcl2 expression was both ER and PR dependent and down regulation of Bcl2 in IDC was significantly correlated with poor prognostic factors.

We studied Fas/FasL, Bcl-2, and M30 CytoDeath in five cases of normal thymus and 41 cases of thymic epithelial tumors (TETs). In normal thymus, Fas was expressed in all epithelial cells, but not in thymic lymphocytes; FasL was weakly expressed only in medullary epithelium and Hassal's corpuscles. In TETs, Fas was expressed in all epithelial cells and lymphocytes, while FasL was differently expressed in epithelial cells of different subtypes of TETs, i.e., type A (2/2), AB (12/12), B1 (0/9), B1/B2 (0/3), B2 (0/1), B2/B3 (1/3), B3 (6/10) and C (1/1), but not in lymphocytes. Bcl2 protein was strongly expressed in medullary-derived lymphocytes of normal thymus and TETs, and weakly expressed only in medullary (spindle) epithelium of TETs. On M30 CytoDeath immunostaining, apoptotic indices were very low in all TETs (0-1.2). In conclusion, since FasL was expressed on the epithelial cells of lymphocyte-depleted (LD) areas of type B2/B3 and/or LD subtypes (type A, AB, B3, C), FasL expression could be relevant for the intracytoplasmic processes of T-cell selection and the regulation of the lymphoid cells inside the tumor, at least partly. However, tumorigenesis of TETs is not necessarily induced by abrogation of apoptosis.

BACKGROUND: The level of proliferation activity is a strong prognostic or predictive indicator in breast cancer, but its optimal measurement is still in debate, necessitating new proliferation markers. In the present study, the prognostic significance of the CKAP2-positive cell count (CPCC), a new proliferation marker, was evaluated, and the results were compared with those for the mitotic activity index (MAI). METHODS: This study included 375 early-stage breast cancer samples collected from two institutions between 2000 and 2006. Immunohistochemical staining was performed using a CKAP2 monoclonal antibody. Cox proportional hazard regression models were fitted to determine the association between the CPCC and relapse-free survival (RFS) amongst three groups formed on the basis of the CPCC or MAI value: groups 2 and 3 showing the middle and highest values, respectively, and group 1 the lowest. RESULTS: After adjustment for age, T stage, N stage, HER2 status, estrogen receptor status, progesterone receptor status, institution, and year of surgical resection, the CPCC was associated with a significantly worse RFS {hazard ratio [HR] = 4.10 (95% CI: 1.64-10.29) for group 2; HR = 4.35 (95% CI: 2.04-10.35) for group 3}. Moreover, its prognostic significance was similar to or higher than that based on the MAI {HR = 2.05 (95% CI: 0.94-4.65) for group 2; HR = 2.35 (95% CI: 1.09-5.10) for group 3}. In subgroup analyses, the CPCC showed a prognostic significance in the luminal A and triple-negative subgroups, but not in the HER2-positive subgroup. CONCLUSIONS: Chromatin CKAP2 is an independent prognostic marker for RFS in early-stage breast cancer, and could potentially replace the MAI in clinical evaluation of proliferation activity. Additionally, our study results suggest that the prognostic significance of proliferation activity differs among the various subgroups of breast cancer.

We performed the immunohistochemical staining for six G1 check point cell cycle proteins to study their expression patterns and roles in the gastric carcinogenesis. We studied 76 cases of paraffin blocks that included the sections of 18 tubular adenomas (TA), 38 early gastric carcinomas (EGC) (20 cases of mucosal type, nine cases of submucosal type with no nodal metastasis, nine cases of submucosal type with nodal metastasis), 20 advanced gastric carcinomas (AGC) (ten cases with no nodal metastasis, ten cases with nodal metastasis). We found that abnormal expression of p16 and p27 increased with the progression of tubular adenomas to advanced gastric cancers. Inverse relationship between pRb and p16 proteins was found in a small portion of the gastric tumors. Expressions of pRb and cdk4 were consistently high in benign and malignant gastric tumors. Expression of cyclin D1 and cyclin E rather decreased with the tumor progression. In conclusion, losses of p16 and p27 seem to play a significant role during the gastric carcinogenesis, and the G1 checkpoint cell cycle proteins such as pRb, cdk4, cyclin D1, and cyclin E variably participate in the gastric carcinogenesis and metastasis by the mechanisms which are yet unknown; thus, further studies need to be performed to elucidate the mechanisms.

Aims/Background: In hepatitis B virus-related hepatocellular carcinoma(HCC), at least 20–40 years of continuous necro-inflammation is necessary for the hepato-carcinogenesis to occur. However, HCC in childhood shows an unusually short latent period and rapid progression. In our previous report, mutation of c-met was found only in childhood HCC, but not in adult HCC. In order to determine the specific biological tumorous features of childhood HCC, a comparison study of childhood and adult HCC was performed. Methods: Eighteen cases of HBV positive HCC (nine children and nine adults aged more than 40 years) were selected. The expression of G1 phase regulatory proteins (cyclin D1, cyclin E and cdk4) was studied using immunohistochemical methods. Loss of heterozygosity (LOH) on chromosomal arms 8p, 13q and 17p was analyzed. Results: Cyclin D1 expression was significantly lower in childhood HCC than in adult HCC (cases of cyclin D1 expression under 3+: childhood 5/9 vs. adult 1/9, p=0.046). No difference in cyclin E and cdk4 expression was found between childhood and adult HCC. LOH frequency on 13q was relatively higher in childhood than in adult HCC (66.7% vs. 22.2%, p=0.058). LOH frequency on 8p and 17p was 44.4% and 33.3% in childhood HCC and 44.4% and 75% in adult HCC with no statistical significance between the two groups. Conclusion: Our data suggest that childhood HCC may be less dependent on cyclin D1 protein for tumor growth and progression than adult HCC, and that the LOH on 13q may be an important feature of childhood HCC.

In proteins, functional divergence involves mutations that modify structure and dynamics. Here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral GFP-like proteins, and their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The photoconversion-competent (red) phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. The mutational sites appear allosterically coupled to the dampening region, while providing conformational mobility to active site residues via epistasis.We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to provide red color. Photoconversion rate measurements provide a bell-shaped curve, indicating that the reaction is controlled by the two apparent pKa values 4.5 (± 0.2) and 7.5 (± 0.2) flanking the chromophore pKa of 6.3 (± 0.1). We tentatively assign these values to the salt-bridged residues Glu222(211) and His203(193), and suggest that reverse protonation may enhance light-induced active site remodeling. In combination, the crystallographic, dynamic and kinetic data support a mechanism that utilizes light to coordinate the transient enhancement of Glu222 proton affinity and His65(63) alpha-carbon acidity, suggesting a concerted process of proton abstraction and main-chain bond scission. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities. (This work was supported by NSF Grant No. MCB-0615938 to R. M. W. and NIH Grant No. U54 GM094599 to R. M. W. and S. B. O.).