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Antimicrobial resistance is a global public health threat. Metallo-β-lactamases (MBLs) inactivate β-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1–B3). We report design, synthesis, and evaluation of BTZ analogues. Structure–activity relationships identified the BTZ thiol as essential, while carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency toward B1 MBLs. These data underscore BTZs as versatile, potent broad-spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors) and provide a rational approach to further improve the tricyclic BBH scaffold.

Persistent infection with high-risk human papillomaviruses (HPVs) is the causal factor in multiple human malignancies, including >99% of cervical cancers and a growing proportion of oropharyngeal cancers. Prolonged expression of the viral oncoproteins E6 and E7 is necessary for transformation to occur. Although some of the mechanisms by which these oncoproteins contribute to carcinogenesis are well-characterised, a comprehensive understanding of the signalling pathways manipulated by HPV is lacking. Here, we present the first evidence to our knowledge that the targeting of a host ion channel by HPV can contribute to cervical carcinogenesis. Through the use of pharmacological activators and inhibitors of ATP-sensitive potassium ion (KATP) channels, we demonstrate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression. Further, expression of SUR1, which forms the regulatory subunit of the multimeric channel complex, was found to be upregulated in both HPV+ cervical cancer cells and in samples from patients with cervical disease, in a manner dependent on the E7 oncoprotein. Importantly, knockdown of SUR1 expression or KATPchannel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. This was confirmed both in vitro and in in vivo tumourigenicity assays. Mechanistically, we propose that the pro-proliferative effect of KATPchannels is mediated via the activation of a MAPK/AP-1 signalling axis. A complete characterisation of the role of KATPchannels in HPV-associated cancer is now warranted in order to determine whether the licensed and clinically available inhibitors of these channels could constitute a potential novel therapy in the treatment of HPV-driven cervical cancer.

Microorganisms do not work alone but instead function as collaborative microsocieties. The spatial distribution of different bacterial strains (micro-biogeography) in a shared volumetric space and their degree of intimacy greatly influences their societal behavior. Current microbiological techniques are commonly focused on the culture of well-mixed bacterial communities and fail to reproduce the micro-biogeography of polybacterial societies. Here, we bioprinted fine-scale bacterial microcosms using chaotic flows induced by a printhead containing a static mixer. This straightforward approach (i.e., continuous chaotic bacterial bioprinting) enables the fabrication of hydrogel constructs with intercalated layers of bacterial strains. These multilayered constructs are used to analyze how the spatial distributions of bacteria affect their social behavior. For example, we show that bacteria within these biological microsystems engage in either cooperation or competition, depending on the degree of shared interface. The extent of inhibition in predator–prey scenarios (i.e., probiotic–pathogen bacteria) increases when bacteria are in greater intimacy. Furthermore, two Escherichia colistrains exhibit competitive behavior in well-mixed microenvironments, whereas stable coexistence prevails for longer times in spatially structured communities. We anticipate that chaotic bioprinting will contribute to the development of a greater complexity of polybacterial microsystems, tissue-microbiota models, and biomanufactured materials.

ABSTRACTIntroduction:Prolonged ozone exposure can produce a state of oxidative stress, which in turn causes alterations in the dynamics of the brain and affects memory and learning. Moreover, different investigations have shown that high flavonoid content berries show a great antioxidant activity. The relationship between the protective effect of the maqui berry extract and its antioxidant properties in the brain has not been studied in depth. Objectives:The present study evaluated whether the protection exerted by the aqueous extract of maqui berry in brain regions associated with cognitive performance is due to its antioxidant capacity. Methods:Sprague Dawley rats were exposed to 0.25 ppm ozone and administered with maqui berry extracts. At the end of the treatments, spatial learning and short- and long-term memory were evaluated, as well as oxidative stress markers. Results:The administration of 50 and 100 mg/kg of the aqueous extract of maqui berry was effective in preventing the cognitive deficit caused by chronic exposure to ozone. The antioxidant effect of the administration of maqui berry was analyzed in the prefrontal cortex, hippocampus, and amygdala. Oxidative stress markers levels decreased and the enzymatic activity of superoxide dismutase diminished in animals exposed to ozone treated with the 50 mg/kg dose of maqui berry. Discussion:These results show a relationship between protection at the cognitive level and a decrease in oxidative stress markers, which suggests that the prevention of cognitive damage is due to the antioxidant activity of the maqui berry.

El cateterismo de senos petrosos inferiores (CSPI) está indicado en el diagnóstico de síndrome de Cushing (SC) hormona adrenocorticotropa (ACTH) dependiente, especialmente cuando los resultados del resto de las pruebas diagnósticas son discordantes.

Human papillomaviruses (HPV) are a major cause of malignancy worldwide, contributing to ~5% of all human cancers including almost all cases of cervical cancer and a growing number of ano-genital and oral cancers. HPV-induced malignancy is primarily driven by the viral oncogenes, E6 and E7, which manipulate host cellular pathways to increase cell proliferation and enhance cell survival, ultimately predisposing infected cells to malignant transformation. Consequently, a more detailed understanding of viral-host interactions in HPV-associated disease offers the potential to identify novel therapeutic targets. Here, we identify that the c-Jun N-terminal kinase (JNK) signalling pathway is activated in cervical disease and in cervical cancer. The HPV E6 oncogene induces JNK1/2 phosphorylation in a manner that requires the E6 PDZ binding motif. We show that blockade of JNK1/2 signalling using small molecule inhibitors, or knockdown of the canonical JNK substrate c-Jun, reduces cell proliferation and induces apoptosis in cervical cancer cells. We further demonstrate that this phenotype is at least partially driven by JNK-dependent activation of EGFR signalling via increased expression of EGFR and the EGFR ligands EGF and HB-EGF. JNK/c-Jun signalling promoted the invasive potential of cervical cancer cells and was required for the expression of the epithelial to mesenchymal transition (EMT)-associated transcription factor Slug and the mesenchymal marker Vimentin. Furthermore, JNK/c-Jun signalling is required for the constitutive expression of HPV E6 and E7, which are essential for cervical cancer cell growth and survival. Together, these data demonstrate a positive feedback loop between the EGFR signalling pathway and HPV E6/E7 expression, identifying a regulatory mechanism in which HPV drives EGFR signalling to promote proliferation, survival and EMT. Thus, our study has identified a novel therapeutic target that may be beneficial for the treatment of cervical cancer.

Las enfermedades infiltrativas sistémicas son un grupo de enfermedades relativamente raras que consisten en la infiltración de células o depósito de sustancias en múltiples órganos y sistemas, entre los que se encuentran las glándulas endocrinas. En este artículo se revisan las alteraciones endocrinológicas de las cuatro más importantes a nivel epidemiológico, como son la sarcoidosis, la histiocitosis de células de Langerhans, la hemocromatosis hereditaria y la amiloidosis sistémica. En cada una de ellas se aportarán recomendaciones al endocrinólogo para el estudio hormonal de estos pacientes.

Protein ubiquitination is a critical regulator of cellular homeostasis. Aberrations in the addition or removal of ubiquitin can result in the development of cancer and key components of the ubiquitination machinery serve as oncogenes or tumour suppressors. An emerging target in the development of cancer therapeutics are the deubiquitinase (DUB) enzymes that remove ubiquitin from protein substrates. Whether this class of enzyme plays a role in cervical cancer has not been fully explored. By interrogating the cervical cancer data from the TCGA consortium, we noted that the DUB USP13 is amplified in ~15% of cervical cancer cases. We confirmed that USP13 expression was increased in cervical cancer cell lines, cytology samples from patients with cervical disease and in cervical cancer tissue. Depletion of USP13 inhibited cervical cancer cell proliferation. Mechanistically, USP13 bound to, deubiquitinated and stabilised Mcl-1, a pivotal member of the anti-apoptotic BCL-2 family. Furthermore, reduced Mcl-1 expression partially contributed to the observed proliferative defect in USP13 depleted cells. Importantly, the expression of USP13 and Mcl-1 proteins correlated in cervical cancer tissue. Finally, we demonstrated that depletion of USP13 expression or inhibition of USP13 enzymatic activity increased the sensitivity of cervical cancer cells to the BH3 mimetic inhibitor ABT-263. Together, our data demonstrates that USP13 is a potential oncogene in cervical cancer that functions to stabilise the pro-survival protein Mcl-1, offering a potential therapeutic target for these cancers.

Ectopic differentiated thyroid carcinoma represents less than 1% of all thyroid carcinoma cases, which usually arises along the cervical midline, and (delving into literature) there are scarce reports of such entity in nuclear medicine imaging studies. We present a rare case of a confirmed synchronous, orthotopic, and heterotopic (in lateral ectopic thyroid tissue) differentiated thyroid carcinoma in a patient who was previously misdiagnosed as having a cervical paraganglioma, based on compatible clinical, analytical, and imagenological (both structural and functional) findings.

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus(MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, and shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, which included interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltransferase and the stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associated antibiotic effects, especially the lack of resistance development, probably stem from the compound’s polypharmacology.

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As a result of the extreme conditions that usually occur in Mediterranean climates, the objective of this work is to study the combined and/or separate effects of saline and alkaline stresses in Portulaca oleracea. The study was carried out to determine the nutritional food potential in relation to plant physiological parameters. The results show that alkaline media in which CaCO3was present did not affect growth but exposure to 100 mM NaCl decreased it greatly. Fatty acid content increased under all stress conditions but to a higher extent with salinity; however, the protein content was increased only by alkaline media. The beneficial effect of each stress on P. oleraceais discussed in light of the physiological response, pointing out the suitability of this plant for human nutrition.

ABSTRACTMetallo-beta-lactamases (MBLs) are broad-spectrum, Zn(II)-dependent lactamases able to confer resistance to virtually every β-lactam antibiotic currently available. The large diversity of active-site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3 that is expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica. This MBL is atypical, since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here, we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active-site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono- and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins, and carbapenems. In silicodocking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast to Ser221, which otherwise is conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design and also posit that GOB enzymes have alternative functions.

Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2•–). O2•–reacts at diffusional rates with nitric oxide to yield a potent nitrating species, peroxynitrite anion (ONOO–). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively modified residue. We previously reported a kof ∼1.0 × 105M–1s–1for the reaction of hMnSOD with ONOO–by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO–, including the necessary re-examination of the second-order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The redetermination of kwas performed by competition kinetics utilizing coumarin boronic acid, which reacts with ONOO–at a rate of ∼1 × 106M–1s–1to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a kof ∼1.0 × 105M–1s–1, fully consistent with the direct method. Proteomic analysis indicated that ONOO–, but not other nitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO–with MnIIIto yield MnIVand intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing how MnSOD constitutes an intramitochondrial target for ONOO–and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34.

Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitroproteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroidessp., and identified iron limitation as one of the major challenges in the bladder environment. In vitroproteome analysis of P. aeruginosaand M. morganiiisolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivoand in vitroprotein profiles of P. aeruginosaand M. morganiialso indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosaseems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganiiis able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system.

Temperature plays an important role in the behavior, ecology, physiology, and evolution of reptiles. Herein, we present selected body temperatures on 9 species of Phrynosomaunder laboratory conditions, and note congeneric differences related to altitude and reproductive mode. The results are discussed with those of other phrynosomatid lizards.