Time-resolved cryo-EM of G-protein activation by a GPCR.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.

Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain.

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.

MDsrv: visual sharing and analysis of molecular dynamics simulations.

Molecular dynamics simulation is a proven technique for computing and visualizing the time-resolved motion of macromolecules at atomic resolution. The MDsrv is a tool that streams MD trajectories and displays them interactively in web browsers without requiring advanced skills, facilitating interactive exploration and collaborative visual analysis. We have now enhanced the MDsrv to further simplify the upload and sharing of MD trajectories and improve their online viewing and analysis. With the new instance, the MDsrv simplifies the creation of sessions, which allows the exchange of MD trajectories with preset representations and perspectives. An important innovation is that the MDsrv can now access and visualize trajectories from remote datasets, which greatly expands its applicability and use, as the data no longer needs to be accessible on a local server. In addition, initial analyses such as sequence or structure alignments, distance measurements, or RMSD calculations have been implemented, which optionally support visual analysis. Finally, based on Mol*, MDsrv now provides faster and more efficient visualization of even large trajectories compared to its predecessor tool NGL.

Machine Learning-Based Prediction of Cattle Activity Using Sensor-Based Data.

Livestock monitoring is a task traditionally carried out through direct observation by experienced caretakers. By analyzing its behavior, it is possible to predict to a certain degree events that require human action, such as calving. However, this continuous monitoring is in many cases not feasible. In this work, we propose, develop and evaluate the accuracy of intelligent algorithms that operate on data obtained by low-cost sensors to determine the state of the animal in the terms used by the caregivers (grazing, ruminating, walking, etc.). The best results have been obtained using aggregations and averages of the time series with support vector classifiers and tree-based ensembles, reaching accuracies of 57% for the general behavior problem (4 classes) and 85% for the standing behavior problem (2 classes). This is a preliminary step to the realization of event-specific predictions.

Mucinous adenocarcinoma arising from complex perianal fistula: a diagnostic and therapeutic challenge.

We present a case of a 64-year-old male with a history of perianal abscesses that have been surgically treated on 10 occasions. Eight months after the last drainage procedure, he presented with a new abscess. Drainage was performed, revealing a cavity with smooth walls, a chronic appearance, filled with mucoid material. An internal fistulous opening was identified at the 6 o'clock position above the anorectal line, which communicated with the described cavity, forming a trans-sphincteric fistula to the mid-anal canal. Biopsy with pathological anatomy showed a mucinous adenocarcinoma with possible intestinal origin (CK20+, CDX2+, TTF1-, CK7+). After completing the evaluation, he was diagnosed with T4N1M0 rectal neoplasia. A diverting colostomy was performed, followed by neoadjuvant therapy, and subsequently, a laparoscopic abdominoperineal amputation. Pathological anatomy revealed residual adenocarcinoma ypT2N0 N0V0L0, R0. This case is notable for both the rarity of a mucinous adenocarcinoma originating in a perianal fistula and the nonspecific clinical presentation of such tumors. Clinical suspicion is crucial, especially in cases of recurrent abscesses with the discharge of mucoid material through fistulous openings, prompting the need for biopsies to ensure proper diagnosis and subsequent optimal treatment.

Prostate gland metastasis as a late relapse of rectal adenocarcinoma.

We present a case report of a 49 year old patient with a known history of rectal adenocarcinoma treated with extended abdominal perineal resection. After five the patient was diagnosed with metastases to the prostate gland. This case stands out not only due to the infrequency of an onset of a colorectal tumor in the prostatic gland but also the late onset of the tumor. In these cases the differential diagnosis between a metastatic tumor and a primary urologic tumor is difficult due to similar morphology and histology, making the CDX-2 immunohistochemical maker expression an important tool to define the histopathology of the tumor.

Lmo2 expression defines tumor cell identity during T-cell leukemogenesis.

The impact of LMO2 expression on cell lineage decisions during T-cell leukemogenesis remains largely elusive. Using genetic lineage tracing, we have explored the potential of LMO2 in dictating a T-cell malignant phenotype. We first initiated LMO2 expression in hematopoietic stem/progenitor cells and maintained its expression in all hematopoietic cells. These mice develop exclusively aggressive human-like T-ALL In order to uncover a potential exclusive reprogramming effect of LMO2 in murine hematopoietic stem/progenitor cells, we next showed that transient LMO2 expression is sufficient for oncogenic function and induction of T-ALL The resulting T-ALLs lacked LMO2 and its target-gene expression, and histologically, transcriptionally, and genetically similar to human LMO2-driven T-ALL We next found that during T-ALL development, secondary genomic alterations take place within the thymus. However, the permissiveness for development of T-ALL seems to be associated with wider windows of differentiation than previously appreciated. Restricted Cre-mediated activation of Lmo2 at different stages of B-cell development induces systematically and unexpectedly T-ALL that closely resembled those of their natural counterparts. Together, these results provide a novel paradigm for the generation of tumor T cells through reprogramming in vivo and could be relevant to improve the response of T-ALL to current therapies.

Obstructive jaundice secondary to a pseudoaneurysm of the superior mesenteric artery.

Superior mesenteric arteries pseudoaneurysms are rare entity, usually asymptomatic but they can appear as an abdominal pain, throbbing mass or shock, being jaundice an exceptional type of presentation. Diagnosis is made by imaging tests (CT) and currently the endovascular approach is the most widespread, reserving open surgery in selected cases. We describe the case of a patient with an 86mm SMA pseudoaneurysm with compression of the extrahepatic bile duct.

Cytomegalovirus colitis seems to be colon cancer. HIV debut.

Cytomegalovirus (CMV) infection is very common in immunosuppressed patients. It can y puede afectar a todo el tracto gastrointestinal, presentándose como úlceras o pseudotumores. A 43-year-old male with no personal background of interest, was studied due to constitutional syndrome. The diagnosis was neoplasia of the right colon, reported by colonoscopy and CT scan. A right hemicolectomy was performed with oncologic character. The definitive histology was CMV infectious colitis with positive immunohistochemical staining. Treatment with ganciclovir was started and the patient was diagnosed with HIV infection. The unusual finding of CMV infection as a pseudotumor can simulate, clinically and radiologically, a colonic neoplasm. It has been described in the literature in patients immunocompromised by HIV; however, the absence of risk factors means that it can be confused with a primary neoformative process.

Sigmoid colon Schwannoma simulating colon cancer.

Schwannomas tumours are uncommonly developed in the gastrointestinal tract (2-6%), located in 12% of cases in small and large intestines. An 87-year-old woman was studied due to epigastric pain and dyspepsia. CT scan and colonoscopy showed a neoplastic process in the sigmoid colon. It was performed an oncologic laparoscopic sigmoidectomy. Histological study described a schwannoma and a positive immunohistochemistry to S-100. The diagnostic challenge is that this is a submucosa lesion, therefore, endoscopy biopsy is realized only in the mucosa. This becomes the differential diagnostic very difficult in order to differentiate from another mesenchymal tumor (GIST or leiomyoma). The immunohistochemistry helps in the diagnostic if it is positive to S- 100 and negative to C-KIT, CD-34, actine and desmine (findings of GIST tumors and leiomyoma). In conclusion, schwannoma diagnostic is tough. Those are asymptomatic tumors with nonspecific radiological findings. Diagnostic confirmation is a S-100 positive immunohistochemistry in the histological study.

Deepint.net: A Rapid Deployment Platform for Smart Territories.

This paper presents an efficient cyberphysical platform for the smart management of smart territories. It is efficient because it facilitates the implementation of data acquisition and data management methods, as well as data representation and dashboard configuration. The platform allows for the use of any type of data source, ranging from the measurements of a multi-functional IoT sensing devices to relational and non-relational databases. It is also smart because it incorporates a complete artificial intelligence suit for data analysis; it includes techniques for data classification, clustering, forecasting, optimization, visualization, etc. It is also compatible with the edge computing concept, allowing for the distribution of intelligence and the use of intelligent sensors. The concept of smart cities is evolving and adapting to new applications; the trend to create intelligent neighbourhoods, districts or territories is becoming increasingly popular, as opposed to the previous approach of managing an entire megacity. In this paper, the platform is presented, and its architecture and functionalities are described. Moreover, its operation has been validated in a case study where the bike renting service of Paris-Vélib' Métropole has been managed. This platform could enable smart territories to develop adapted knowledge management systems, adapt them to new requirements and to use multiple types of data, and execute efficient computational and artificial intelligence algorithms. The platform optimizes the decisions taken by human experts through explainable artificial intelligence models that obtain data from IoT sensors, databases, the Internet, etc. The global intelligence of the platform could potentially coordinate its decision-making processes with intelligent nodes installed in the edge, which would use the most advanced data processing techniques.

Analysis of polysaccharide hydrolases secreted by Aspergillus flavipes FP-500 on corn cobs and wheat bran as complex carbon sources.

Aspergillus flavipes FP-500 is a Mexican native strain that has been reported as a good producer of xylanases and pectinases; therefore, it promises a strong impact on biotechnology. To provide an overview of protein secretion by A. flavipes, we carried out a comparative proteome analysis of extracellular proteins in liquid cultures with two heterogeneous agro-industrial residues; corn cob (CC) and wheat bran (WB), as carbon sources. Extracellular proteins obtained from both cultures were identified using MS/MS spectrometry. We identified 134 proteins, which were classified into four groups: glycosyl hydrolases (GH), esterases/proteases, miscellaneous proteins, and unidentified proteins. Around 50% of the total proteins identified were GH such as xylanases, ß-xylosidases, ß-galactosidases, cellulolytic enzymes like ß-glucosidase, endoglucanases, and cellobiohydrolases. From this family, a core of 22 (16%) of the proteins identified were found in both substrates, CC and WB, whereas 30% and 54% were unique for CC and WB, respectively. In the esterases/proteases group, proteases, lipases and esterases like feruloylesterases, and acetyl-xylanesterase were identified. Proteins with diverse functions such as monophosphate dehydrogenase or N-acetylglucosaminidase were present. Here, we present strong evidences indicating that the composition and heterogeneity of the used carbon source determine the specific set of protein secreted by the fungus.

Crebbp loss cooperates with Bcl2 overexpression to promote lymphoma in mice.

CREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 overexpression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B cells, we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, and showed Myc binding. Through the analysis of CREBBP mutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these 2 diseases, with higher frequencies of nonsense/frameshift mutations in DLBCL compared with FL. Together, our data therefore provide important links between Crebbp inactivation and Bcl2 dependence and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.

Novel ETV6-RUNX1 Mouse Model to Study the Role of ELF-MF in Childhood B-Acute Lymphoblastic Leukemia: a Pilot Study.

Exposure to extremely low-frequency magnetic fields (ELF-MFs) has been classified by the International Agency for Research on Cancer (IARC) as "possibly carcinogenic to humans," based on limited scientific evidence concerning childhood leukemia. This assessment emphasized the lack of appropriate animal models recapitulating the natural history of this disease. Childhood B-cell acute lymphoblastic leukemia (B-ALL) is the result of complex interactions between genetic susceptibility and exposure to exogenous agents. The most common chromosomal alteration is the ETV6-RUNX1 fusion gene, which confers a low risk of developing the malignancy by originating a preleukemic clone requiring secondary hits for full-blown disease to appear. To develop potential prophylactic interventions, we need to identify the environmental triggers of the second hit. Recently, we generated a B-ALL mouse model of the human ETV6-RUNX1+ preleukemic state. Here, we present the results from the ARIMMORA pilot study, obtained by exposing 34 Sca1-ETV6-RUNX1 mice (vs. 27 unexposed) to a 50 Hz magnetic field of 1.5 mT with both fundamental and harmonic content, with an on/off cycle of 10 min/5 min, for 20 h/day, from conception until 3 months of age. Mice were monitored until 2 years of age and peripheral blood was periodically analyzed by flow cytometry. One of the exposed mice developed B-ALL while none of the non-exposed did. Although the results are statistically non-significant due to the limited number of mice used in this pilot experiment, overall, the results show that the newly developed Sca1-ETV6-RUNX1 mouse can be successfully used for ELF-MF exposure studies about the etiology of childhood B-ALL. Bioelectromagnetics. 2019;40:343-353. © 2019 Bioelectromagnetics Society.

Annexin A1, Annexin A2, and Dyrk 1B are upregulated during GAS1-induced cell cycle arrest.

GAS1 is a pleiotropic protein that has been investigated because of its ability to induce cell proliferation, cell arrest, and apoptosis, depending on the cellular or the physiological context in which it is expressed. At this point, we have information about the molecular mechanisms by which GAS1 induces proliferation and apoptosis; but very few studies have been focused on elucidating the mechanisms by which GAS1 induces cell arrest. With the aim of expanding our knowledge on this subject, we first focused our research on finding proteins that were preferentially expressed in cells arrested by serum deprivation. By using a proteomics approach and mass spectrometry analysis, we identified 17 proteins in the 2-DE protein profile of serum deprived NIH3T3 cells. Among them, Annexin A1 (Anxa1), Annexin A2 (Anxa2), dual specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1B), and Eukaryotic translation initiation factor 3, F (eIf3f) were upregulated at transcriptional the level in proliferative NIH3T3 cells. Moreover, we demonstrated that Anxa1, Anxa2, and Dyrk1b are upregulated at both the transcriptional and translational levels by the overexpression of GAS1. Thus, our results suggest that the upregulation of Anxa1, Anxa2, and Dyrk1b could be related to the ability of GAS1 to induce cell arrest and maintain cell viability. Finally, we provided further evidence showing that GAS1 through Dyrk 1B leads not only to the arrest of NIH3T3 cells but also maintains cell viability.

Cor interacts with outer membrane proteins to exclude FhuA-dependent phages.

Superinfection exclusion (Sie) of FhuA-dependent phages is carried out by Cor in the Escherichia coli mEp167 prophage lysogenic strain. In this work, we present evidence that Cor is an outer membrane (OM) lipoprotein that requires the participation of additional outer membrane proteins (OMPs) to exclude FhuA-dependent phages. Two Cor species of ~13 and ~8.5 kDa, corresponding to the preprolipoprotein/prolipoprotein and lipoprotein, were observed by Western blot. Cell mutants for CorC17F, CorA18D and CorA57E lost the Sie phenotype for FhuA-dependent phages. A copurification affinity binding assay combined with LC_ESI_MS/MS showed that Cor bound to OMPs: OmpA, OmpC, OmpF, OmpW, LamB, and Slp. Interestingly, Sie for FhuA-dependent phages was reduced on Cor overexpressing FhuA+ mutant strains, where ompA, ompC, ompF, ompW, lamB, fhuE, genes were knocked out. The exclusion was restored when these strains were supplemented with plasmids expressing these genes. Sie was not lost in other Cor overexpressing FhuA+ null mutant strains JW3938(btuB-), JW5100(tolB-), JW3474(slp-). These results indicate that Cor interacts and requires some OMPs to exclude FhuA-dependent phages.

The Making of Leukemia.

Due to the clonal nature of human leukemia evolution, all leukemic cells carry the same leukemia-initiating genetic lesions, independently of the intrinsic tumoral cellular heterogeneity. However, the latest findings have shown that the mode of action of oncogenes is not homogeneous throughout the developmental history of leukemia. Studies on different types of hematopoietic tumors have shown that the contribution of oncogenes to leukemia is mainly mediated through the epigenetic reprogramming of the leukemia-initiating target cell. This driving of cancer by a malignant epigenetic stem cell rewiring is, however, not exclusive of the hematopoietic system, but rather represents a common tumoral mechanism that is also at work in epithelial tumors. Tumoral epigenetic reprogramming is therefore a new type of interaction between genes and their target cells, in which the action of the oncogene modifies the epigenome to prime leukemia development by establishing a new pathological tumoral cellular identity. This reprogramming may remain latent until it is triggered by either endogenous or environmental stimuli. This new view on the making of leukemia not only reveals a novel function for oncogenes, but also provides evidence for a previously unconsidered model of leukemogenesis, in which the programming of the leukemia cellular identity has already occurred at the level of stem cells, therefore showing a role for oncogenes in the timing of leukemia initiation.

Structural and kinetics characterization of the F1F0-ATP synthase dimer. New repercussion of monomer-monomer contact.

Ustilago maydis is an aerobic basidiomycete that fully depends on oxidative phosphorylation for its supply of ATP, pointing to mitochondria as a key player in the energy metabolism of this organism. Mitochondrial F1F0-ATP synthase occurs in supramolecular structures. In this work, we isolated the monomer (640kDa) and the dimer (1280kDa) and characterized their subunit composition and kinetics of ATP hydrolysis. Mass spectrometry revealed that dimerizing subunits e and g were present in the dimer but not in the monomer. Analysis of the ATPase activity showed that both oligomers had Michaelis-Menten kinetics, but the dimer was 7 times more active than the monomer, while affinities were similar. The dimer was more sensitive to oligomycin inhibition, with a Ki of 24nM, while the monomer had a Ki of 169nM. The results suggest that the interphase between the monomers in the dimer state affects the catalytic efficiency of the enzyme and its sensitivity to inhibitors.

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria.

The ζ subunit is a novel inhibitor of the F1FO-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF1) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F1-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599-608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F1-ATPase and F1-ζ models of P. denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F1-ATPase at the open catalytic αE/ßE interface, and two partial γ rotations lock the N terminus of ζ in an "inhibition-general core region," blocking further γ rotation, while the ζ globular domain anchors it to the closed αDP/ßDP interface. Heterologous inhibition of the F1-ATPase of P. denitrificans by the mitochondrial IF1 supported both the modeled ζ binding site at the αDP/ßDP/γ interface and the endosymbiotic α-proteobacterial origin of mitochondria. In summary, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF1 or the bacterial ϵ binds. The proposed pawl mechanism is coupled to the rotation of the central γ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the α-proteobacteria.

Rectal inflammatory stenosis secondary to Chlamydia trachomatis: a case report.

The rectal inflammatory originated strictures constitute a rare cause of intestinal obstruction. We present a 30-year-old male patient with a history of HIV and protctitis caused by Chalmydia trachomatis and HSV-2, in which develops a low intestinal obstruction refractory to medical treatment. Surgery was performed with good clinical evolution. The medical treatment constitutes the fundamental basis of the therapy in these patients. Despite that, when fibrotic stenoses are not treatable medical or endoscopically, they often require surgical treatment. We must pay attention to the proctitis infectious diseases as a cause of rectal stenosis, especially by Chlamydia trachomatis, and assess surgical option in refractory cases.