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Objective: According to the 2017 World Health Organization classification of neuro-endocrine tumors, pituitary adenomas (PAs) are classified according to immunoexpression of the pituitary-specific transcription factors (TFs). A small subset of PAs exhibit multiple TF staining on immunohistochemistry and we present a series of 27 pathologically-confirmed cases of dual TF staining PAs (dsTF-PAs), and report clinically relevant implications., Methods: A retrospective chart review of a multi-institutional database of patients with PAs surgically resected between 2008-2021 was performed. PAs expressing immunopositivity 2+ TFs. Patient demographics, neuro-imaging characteristics, histopathologic findings, and clinical data were collected., Results: Twenty-seven patients had pathologically verified dsTF-PAs, of whom 17 were female (63%), with ages ranging from 20-84 years. Twenty-three (85.2%) patients harbored functional PAs, with acromegaly being the most common functional subtype (86.4%). The most common combination of TFs within a single tumor was PIT-1/SF-1 (85.2%). Six PAs exhibited Knosp cavernous sinus invasion grades of 3 or 4 and the Ki-67 labeling index was ≥3% in 6 patients (24.0%) and all stained for PIT-1/SF-1. Hormonal remission was achieved in 78% of functional dsTF-PAs. No PAs showed evidence of recurrence or progression over the mean follow-up period of 28.5 months., Conclusions: PAs exhibiting dsTF-PAs represent a small but clinically relevant diagnostic subset of PAs according to the 2021 World Health Organization criteria, as a majority are GH-producing. Precise classification using TF staining plays a key role in understanding the biology of these tumors. Favorable outcomes can be achieved in this subset of PAs with evolving TF classification., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The direct endoscopic endonasal approach (EEA) has become the primary technique used for resection of sellar pathology, meriting investigation into the risk factors for complications and predictors of postoperative outcomes after direct EEA., Methods: We retrospectively analyzed the patient and tumor characteristics from 404 patients who had undergone direct EEA for sellar pathology at the USC Pituitary Center from September 2011 to December 2019., Results: Of the 404 pathologic entities included, 349 (86%) were pituitary adenomas (PAs), 29 (7%) were Rathke cleft cysts, and 26 (6%) were other sellar lesions. The mean lesion diameter was 2.3 cm, with 34 microadenomas (10%) and 315 macroadenomas (90%). Cavernous sinus invasion was present in 39% of the PAs. No patient died. The surgical complications included internal carotid artery injury without neurological sequelae (0.2%), vision loss (0.7%), meningitis (0.7%), cerebrospinal fluid leak (4%), epistaxis (4%), sinusitis (1%), transient cranial nerve paresis (0.5%), and postoperative abscess (0.25%). New hypopituitarism developed in 3%. Gross total resection was achieved in 208 PA cases (58%). Clinical improvement of headaches and visual deficits were reported for 67% and 76% of cases, respectively. Hormonal remission was achieved in 82% of patients with functional PAs. The median hospital stay was 2 days, with 34 patients (8%) readmitted within 30 days and 10 (3%) undergoing early reoperation. Disease recurrence or progression developed in 10% and was less likely in the case of gross total resection and apoplexy., Conclusion: In the present, large, consecutive, mostly single-surgeon series, the patients experienced clinical improvement in most preoperative symptoms and had low rates of perioperative morbidity. We have demonstrated that direct EEA can be efficiently, safely, and successfully performed by a neurosurgical team., (Copyright © 2021 Elsevier Inc. All rights reserved.)

V-ATPases are ubiquitous proton-transporting ATPases of eukaryotic and prokaryotic membranes that utilize energy from ATP hydrolysis. The hydrophilic catalytic part called V 1 -ATPase is composed of a ring-shaped hexametric A 3 B 3 complex and a central DF shaft. We previously proposed a rotation mechanism of the Enterococcus hirae V 1 -ATPase based on the crystal structures of the V 1 and A 3 B 3 complexes. However, the driving force that induces the conformational changes of A 3 B 3 and rotation of the DF shaft remains unclear. In this study, we investigated the binding affinity changes between subunits of V 1 -ATPase by surface plasmon resonance analysis. The binding of ATP to subunit A was found to considerably increase the affinity between the A and B subunits, and thereby ATP binding contributes to forming the A 1 B 1 tight conformation. Furthermore, the DF shaft bound to the reconstituted A 1 B 1 complex with high affinity, suggesting that the tight A 1 B 1 complex is a major binding unit of the shaft in the A 3 B 3 ring complex. Based on these results, we propose that rotation of the V 1 -ATPase is driven by affinity changes between each subunit via thermal fluctuations., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The management of pituitary apoplexy, caused by acute hemorrhage and/or infarction of a pituitary adenoma, is debated., Objective: To analyze clinical characteristics of patients undergoing endoscopic endonasal approaches (EEAs) for pituitary apoplexy., Methods: A retrospective review of patients at our institution from 2012 to 2018 undergoing EEA for pituitary apoplexy diagnosed clinically and with imaging/pathologic findings. Analysis included demographics, symptoms, neuroendocrine deficits, neuroimaging, complications, symptom resolution, and follow-up details., Results: Fifty patients (mean age, 53 years) were included. Preoperative symptoms included headache (86%), vision loss (62%), and cranial nerve paresis (40%). Mean tumor diameter was 2.7 cm and extrasellar extension was observed in 96% of tumors. Twenty-eight tumors were hemorrhagic (76%), 24 were necrotic (65%), and 13 (35%) had both features. Magnetic resonance imaging showed gross total resection in 58% of patients. Headache and vision loss improved in 87% and 86% of presenting patients. Cranial nerve paresis resolved in 72% of patients, partially improved in 11%, and remained unchanged in 17%. There were no deaths or carotid artery injuries. Surgical complications included postoperative cerebrospinal fluid leak (n = 4, 8%), epistaxis (n = 2, 4%), postoperative abscess (n = 1, 2%), and transient postoperative vision loss requiring reoperation (n = 1, 4%). Endocrinopathies improved in 21% of patients and panhypopituitarism persisted in 48% and developed in 6% of patients. Mean follow-up time was 26 months; 2 patients experienced recurrence., Conclusions: EEA for pituitary apoplexy is effective in rapidly improving headache and visual symptoms. Although neuro-ophthalmic deficits often improve over time, panhypopituitarism persists in most patients after surgical resection., (Copyright © 2020. Published by Elsevier Inc.)

When the replisome collapses at a DNA damage site, a sequence-independent replication restart system is required. In Escherichia coli, PriA, PriB, and DnaT assemble in an orderly fashion at the stalled replication fork and achieve the reloading of the replisome. PriB-DnaT interaction is considered a significant step in the replication restart. In this study, we examined the contribution of the residues Ser20, His26 and Ser55, which are located on the PriB dimer interface. These residues are proximal to Glu39 and Arg44, which are important for PriB-DnaT interaction. Mutational analyses revealed that His26 and Ser20 of PriB are important for the interaction with DnaT, and that the Ser55 residue of PriB might have a role in negatively regulating the DnaT binding. These residues are involved in not only the interaction between PriB and DnaT but also the dissociation of single-stranded DNA (ssDNA) from the PriB-ssDNA complex due to DnaT binding. Moreover, NMR study indicates that the region Asp66-Glu76 on the linker between DnaT domains is involved in the interaction with wild-type PriB. These findings provide significant information about the molecular mechanism underlying replication restart in bacteria., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Central neurocytomas comprise nearly half of adult intraventricular neoplasms. The median age of onset is 34 years. It is typically a low-grade neoplasm (World Health Organization grade II), although some cases of malignant neurocytomas have been described. We present a rare case of an atypical central neurocytoma with craniospinal dissemination, including both imaging and pathologic findings., (Published by Elsevier Inc.)

CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4(+) T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-beta-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.

N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand-receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1-10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B(max) value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.

Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein-protein interactions have been performed, the very weak interactions generally observed in cell-cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (K(d) greater than muM range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLA-Cw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 muM (HLA-Cw4) and 5.7 muM (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell-cell recognition receptors involved in immunologically relevant events.

Purpose: PD173074, a small molecule inhibitor of VEGF-RII and FGF-RI, targets neoangiogenesis and mitogenesis. This study aimed to analyze a single-compound-driven inhibition of FGF and VEGF receptors in pancreatic cancer., Experimental Design: RT-PCR and Western blots were performed to quantify protein expression and phosphorylation. Anchorage dependent and independent growth assays were used to study cell growth. With flow cytometry, cell cycle analysis and apoptosis were studied. In vivo HPAF-II and MIA PaCa-2 cells were xenografted. Animals were treated daily for 10 weeks. Immunohistochemistry was used to quantify microvessel density and apoptosis., Results: Highest levels of FGF-RI were detectable in MIA PaCa-2 cells, lowest in HPAF-II cells. PD173074 inhibited cell growth most prominently in cells expressing high levels of FGF-RI. Cell cycle progression was inhibited by blocking transition in the G(0)/G(1) phase, and consequently, apoptosis was increased. In vivo significant inhibition of orthotopic tumor growth was achieved by a combination effect of inhibition of mitogenesis, induction of apoptosis, and reduction of angiogenesis in PD173074-treated animals., Conclusions: These data highlight VEGF-RII and FGF-RI as therapeutic targets and suggest a potential role for the combined use of tyrosine kinase inhibitors in the management of inoperable pancreatic cancer patients.

The human inhibitory receptor, leukocyte immunoglobulin (Ig)-like receptor B1 (also called Ig-like transcript (ILT) 2, CD85j), is broadly expressed on leukocytes. LILRB1 binds to a wide range of major histocompatibility complex class I molecules (MHCIs) and transduces negative signals that can, for example, prevent killing of MHCI-expressing cells. Here we report the kinetic, thermodynamic, NMR and crystallographic analyses of MHCI recognition by LILRB1. Kinetic studies demonstrated that LILRB1 binds to MHCIs with fast association and dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses showed that LILRB1-MHCI interactions are entropically driven (-TdeltaS = -9.4 approximately -6.6 kcal mol(-1)) with low heat capacity changes (deltaC(p) = -0.22 approximately -0.10 kcal mol(-1) K(-1)). The crystal structures of LILRB1 in the different crystal forms exhibited variation in the elbow angle between the two N-terminal Ig-like domains, indicating interdomain flexibility. Consistently, NMR analysis provided the direct evidence of the conformational changes of LILRB1 upon the MHCI binding. These findings suggest that LILRB1-MHCI interactions, while involving some conformational adjustment, are not accompanied by a very large reduction in conformational flexibility at the binding interface. This mode of binding is distinct from "induced-fit" binding, which is associated with large reductions in conformational flexibility, and would be suitable for rapid engagement of MHCIs to enable fast monitoring of the expression level of MHCIs on target cells.

Novel, cell-based assays, based on bioluminescence resonance energy transfer, have been developed for FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling at receptor-proximal steps. In a stable transfectant of the HEK-293 cell line expressing human FcepsilonRIalpha, FcepsilonRIbeta, and FcRgamma-GFP2 and Syk(1-265)-Rluc fusion proteins, FcepsilonRI cross-linking markedly increased BRET2 ratios, which are the ratios of GFP2 emission to Rluc emission. These ratios reflect the FcRgamma-GFP2-Syk(1-265)-Rluc interaction in living cells. The signals are specifically inhibited by the Src-family kinase inhibitor PP2. Separately, in transient transfectants expressing GPVI, FcRgamma-GFP2, and Syk(1-265)-Rluc, the GPVI-specific ligand convulxin induced a two-fold increase in the BRET2 ratio and this increase was also inhibited by PP2. Finally, a differential assay was developed which permits the measurement of FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling in the same cell. These assays provide useful methods for monitoring FcRgamma-Syk interaction in real time in living cells and may contribute to the understanding of signal regulation through FcRgamma-containing receptors.

PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.