IGFBP-2 partly mediates the early metabolic improvements caused by bariatric surgery.

Insulin-like growth factor-binding protein (IGFBP)-2 is a circulating biomarker of cardiometabolic health. Here, we report that circulating IGFBP-2 concentrations robustly increase after different bariatric procedures in humans, reaching higher levels after biliopancreatic diversion with duodenal switch (BPD-DS) than after Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG). This increase is closely associated with insulin sensitization. In mice and rats, BPD-DS and RYGB operations also increase circulating IGFBP-2 levels, which are not affected by SG or caloric restriction. In mice, Igfbp2 deficiency significantly impairs surgery-induced loss in adiposity and early improvement in insulin sensitivity but does not affect long-term enhancement in glucose homeostasis. This study demonstrates that the modulation of circulating IGFBP-2 may play a role in the early improvement of insulin sensitivity and loss of adiposity brought about by bariatric surgery.

Assessment of the biochemical pathways for acetaminophen toxicity: Implications for its carcinogenic hazard potential.

In 2019 California's Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen. In parallel with this review, herein we evaluated the mechanistic data related to the steps and timing of cellular events following therapeutic recommended (≤4 g/day) and higher doses of acetaminophen that may cause hepatotoxicity to evaluate whether these changes indicate that acetaminophen is a carcinogenic hazard. At therapeutic recommended doses, acetaminophen forms limited amounts of N-acetyl-p-benzoquinone-imine (NAPQI) without adverse cellular effects. Following overdoses of acetaminophen, there is potential for more extensive formation of NAPQI and depletion of glutathione, which may result in mitochondrial dysfunction and DNA damage, but only at doses that result in cell death - thus making it implausible for acetaminophen to induce the kind of stable, genetic damage in the nucleus indicative of a genotoxic or carcinogenic hazard in humans. The collective data demonstrate a lack of a plausible mechanism related to carcinogenicity and are consistent with rodent cancer bioassays, epidemiological results reviewed in companion manuscripts in this issue, as well as conclusions of multiple international health authorities.

Edaravone attenuates lung injury in a hind limb ischemia-reperfusion rat model: A histological, immunohistochemical and biochemical study.

Edaravone is a potent free radical scavenger that has a promising role in combating many acute lung injuries. Ischemia/reperfusion process is a serious condition that may lead to multiple organ dysfunctions. This work was designed to investigate novel mechanisms underlying ischemia/reperfusion-induced lung injury and to evaluate the protective role of edaravone. Thirty adult male rats were divided into three experimental groups; operated with no ischemia (Sham-group), ischemia/reperfusion (I/R) group and edaravone-I/R group. Hind limb ischemia was carried out by clamping the femoral artery. After two hours of ischemia for the hind limb, the rat underwent 24h of reperfusion. Rats in the edaravone-I/R group received edaravone (3mg/kg), 30min before induction of ischemia. At the end of the I/R trial, specimens from the lungs were processed for histological, immunohistochemical, enzyme assay, and RT-qPCR studies. Specimens from I/R group showed focal disruption of the alveolar architecture. Extensive mononuclear cellular infiltration particularly with neutrophils and dilated congested blood capillaries were observed. A significant increase in iNOS, NF-κB, and COX-2 immunoreaction was detected and confirmed by RT-qPCR. Ultrastructural examination showed RBCs and fluid inside alveoli, cellular infiltration, and vacuolations of the inter-alveolar septum. In addition to the presence of extravasated neutrophils and RBCs within the inter-alveolar septum. In contrast, minimal changes were observed in rats which received edaravone before the onset of the ischemia. It could be concluded that edaravone exerted a potent protective effect against lung injury induced by a hind limb I/R in rats through its antioxidant and anti-inflammatory activities.

Predicting biomedical relationships using the knowledge and graph embedding cascade model.

Advances in machine learning and deep learning methods, together with the increasing availability of large-scale pharmacological, genomic, and chemical datasets, have created opportunities for identifying potentially useful relationships within biochemical networks. Knowledge embedding models have been found to have value in detecting knowledge-based correlations among entities, but little effort has been made to apply them to networks of biochemical entities. This is because such networks tend to be unbalanced and sparse, and knowledge embedding models do not work well on them. However, to some extent, the shortcomings of knowledge embedding models can be compensated for if they are used in association with graph embedding. In this paper, we combine knowledge embedding and graph embedding to represent biochemical entities and their relations as dense and low-dimensional vectors. We build a cascade learning framework which incorporates semantic features from the knowledge embedding model, and graph features from the graph embedding model, to score the probability of linking. The proposed method performs noticeably better than the models with which it is compared. It predicted links and entities with an accuracy of 93%, and its average hits@10 score has an average of 8.6% absolute improvement compared with original knowledge embedding model, 1.1% to 9.7% absolute improvement compared with other knowledge and graph embedding algorithm. In addition, we designed a meta-path algorithm to detect path relations in the biomedical network. Case studies further verify the value of the proposed model in finding potential relationships between diseases, drugs, genes, treatments, etc. Amongst the findings of the proposed model are the suggestion that VDR (vitamin D receptor) may be linked to prostate cancer. This is backed by evidence from medical databases and published research, supporting the suggestion that our proposed model could be of value to biomedical researchers.

Electrochemical simulation of metabolism for antitumor-active imidazoacridinone C-1311 and in silico prediction of drug metabolic reactions.

The metabolism of antitumor-active 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) has been investigated widely over the last decade but some aspects of molecular mechanisms of its metabolic transformation are still not explained. In the current work, we have reported a direct and rapid analytical tool for better prediction of C-1311 metabolism which is based on electrochemistry (EC) coupled on-line with electrospray ionization mass spectrometry (ESI-MS). Simulation of the oxidative phase I metabolism of the compound was achieved in a simple electrochemical thin-layer cell consisting of three electrodes (ROXY™, Antec Leyden, the Netherlands). We demonstrated that the formation of the products of N-dealkylation reactions can be easily simulated using purely instrumental approach. Newly reported products of oxidative transformations like hydroxylated or oxygenated derivatives become accessible. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass ion data and tandem mass spectrometry experiments. In silico prediction of main sites of C-1311 metabolism was performed using MetaSite software. The compound was evaluated for cytochrome P450 1A2-, 3A4-, and 2D6-mediated reactions. The results obtained by EC were also compared and correlated with those of reported earlier for conventional in vitro enzymatic studies in the presence of liver microsomes and in the model peroxidase system. The in vitro experimental approach and the in silico metabolism findings showed a quite good agreement with the data from EC/ESI-MS analysis. Thus, we conclude here that the electrochemical technique provides the promising platform for the simple evaluation of drug metabolism and the reaction mechanism studies, giving first clues to the metabolic transformation of pharmaceuticals in the human body.

Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1.

Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo and in vitro experiments revealed that, unlike in yeast, Cdk1-mediated phosphorylation of human Cdc14A did not control its catalytic activity but likely modulated its interaction with other proteins in early mitosis. These findings point to differences in Cdk1-mediated mechanisms of regulation between human and yeast Cdc14 orthologues.

Perceived Stress and Surgical Wound Cytokine Patterns.

This was a pilot study to examine pre- and postoperative stress experienced by women who were undergoing autologous breast reconstruction and how stress might impact wound healing, specifically examining cytokines and other chemical mediators in the wound environment. A nonexperimental descriptive design over time was utilized. Participants were women who were undergoing autologous abdominal breast reconstruction for breast cancer (N = 20). Data were collected preoperatively and at 24, 48, 72, and 96 hr postsurgery. Complications were monitored intraoperatively and up to 30 days postsurgery. Psychological stress was measured with the 10-item Perceived Stress Scale (PSS), the Impact of Events Scale-Revised (IES-R), and a 100-mm Visual Analog Scale (VAS). Cytokines were assayed using the 27-plex kit with a Bio-Plex Plus. Although breast cancer is considered a stressor, in this sample of women, scores of the PSS, IES-R, and VAS showed that in fact these participants experienced low levels of psychological stress. All measured biochemical mediators in serum and wound fluid were detected and trends were identified. IL-1ra, IL-6, IL-8, G-CSF, IP-10, MCP-1, MIP-1ß, RANTES, and VEGF were present in the highest concentrations. Significant changes in levels of cytokines in wound fluid were observed in IL-1ß, IL-2, IL-5, IL-6, IL-8, IL-9, IL-10, IL-17, FGF-basic, G-CSF, MIP-1α, PDGF-bb, MIP-1ß, RANTES, and TNF-α. The remaining cytokine concentrations stayed stable over time. These findings suggest that although these women were not experiencing high levels of stress, meaningful cytokine patterns were detected.

Recent Advances in Enzymatic Complexity Generation: Cyclization Reactions.

Enzymes in biosynthetic pathways, especially in plant and microbial metabolism, generate structural and functional group complexity in small molecules by conversion of acyclic frameworks to cyclic scaffolds via short, efficient routes. The distinct chemical logic used by several distinct classes of cyclases, oxidative and non-oxidative, has recently been elucidated by genome mining, heterologous expression, and genetic and mechanistic analyses. These include enzymes performing pericyclic transformations, pyran synthases, tandem acting epoxygenases, and epoxide "hydrolases", as well as oxygenases and radical S-adenosylmethionine enzymes that involve rearrangements of substrate radicals under aerobic or anaerobic conditions.

Positive surgical margins and biochemical recurrence following minimally-invasive radical prostatectomy - An analysis of outcomes from a UK tertiary referral centre.

BACKGROUND: Positive surgical margins are a strong prognostic marker of disease outcome following radical prostatectomy, though prior evidence is largely from a PSA-screened population. We therefore aim to evaluate the biochemical recurrence in men with positive surgical margins (PSM) after minimally-invasive radical prostatectomy (MIRP) in a UK tertiary centre. METHODS: Retrospective study of men undergoing laparoscopic or robotic-assisted radical prostatectomy between 2002 and 2014. Men with positive surgical margins (PSM) were identified and their biochemical recurrence (BCR) rate compared with men without PSM. The primary outcome measures were BCR rates and time to BCR. Cox regression was used to estimate adjusted hazard ratios for biochemical recurrence rate (BCR), accounting for potential confounders. RESULTS: Five hundred ninety-two men were included for analysis. Pre-operative D'Amico risk stratification showed 37.5%, 53.3% and 9.3% of patients in the low, intermediate and high-risk groups, respectively. On final pathological analysis, the proportion of patients with local staging pT2, pT3a and pT3b was 68.8%, 25.2% and 6.1% respectively. Overall positive margin rate was 30.6%. On multivariate analysis, the only pre-operative factor associated with PSM was age >65years. Patients with PSM were more likely to have higher tumour volume and more advanced pathological local stage. The BCR rate was 10.7% in margin-positive patients and 5.1% in margin-negative patients, at median 4.4-year follow-up. Upon multivariate analysis, high pre-operative PSA and high Gleason group were the only significant predictors of BCR (P<0.05). CONCLUSIONS: In comparison to patients with negative surgical margins, those with PSM do not translate into worse medium-term oncological outcomes in the majority of cases amongst our cohort. We found that high pre-operative PSA and high Gleason group were the only significant predictors of BCR.

A Novel Synthetic Pathway Enables Microbial Production of Polyphenols Independent from the Endogenous Aromatic Amino Acid Metabolism.

Numerous plant polyphenols have potential applications as pharmaceuticals or nutraceuticals. Stilbenes and flavonoids as most abundant polyphenols are synthesized from phenylpropanoids, which are exclusively derived from aromatic amino acids in nature. Several microorganisms were engineered for the synthesis of biotechnologically interesting plant polyphenols; however, low activity of heterologous ammonia lyases, linking endogenous microbial aromatic amino acid biosynthesis to phenylpropanoid synthesis, turned out to be the limiting step during microbial synthesis. We here developed an alternative strategy for polyphenol production from cheap benzoic acids by reversal of a ß-oxidative phenylpropanoid degradation pathway avoiding any ammonia lyase activity. The synthetic pathway running in the non-natural direction is feasible with respect to thermodynamics and involved reaction mechanisms. Instantly, product titers of 5 mg/L resveratrol could be achieved in recombinant Corynebacterium glutamicum strains indicating that phenylpropanoid synthesis from 4-hydroxybenzoic acid can in principle be implemented independently from aromatic amino acids and ammonia lyase activity.

CD73 and AMPD3 deficiency enhance metabolic performance via erythrocyte ATP that decreases hemoglobin oxygen affinity.

Erythrocytes are the key target in 5'-AMP induced hypometabolism. To understand how regulation of endogenous erythrocyte AMP levels modulates systemic metabolism, we generated mice deficient in both CD73 and AMPD3, the key catabolic enzymes for extracellular and intra-erythrocyte AMP, respectively. Under physiological conditions, these mice displayed enhanced capacity for physical activity accompanied by significantly higher food and oxygen consumption, compared to wild type mice. Erythrocytes from Ampd3(-/-) mice exhibited higher half-saturation pressure of oxygen (p50) and about 3-fold higher levels of ATP and ADP, while they maintained normal 2,3-bisphosphoglycerate (2,3-BPG), methemoglobin levels and intracellular pH. The affinity of mammalian hemoglobin for oxygen is thought to be regulated primarily by 2,3-BPG levels and pH (the Bohr effect). However, our results show that increased endogenous levels of ATP and ADP, but not AMP, directly increase the p50 value of hemoglobin. Additionally, the rise in erythrocyte p50 directly correlates with an enhanced capability of systemic metabolism.

A theoretical model of biochemical control engineering based on the relation between oestrogens/progestagens and prostaglandins.

A biological complex organism is involuntarily guided from all sides by measure and regulation systems. The human being is such a complex organism. Many cyclical processes are simultaneously at work, making it unclear how and why which process takes place at which moment. Noticeable examples are the 28-day menstrual cycle and the 40-week pregnancy. The time of activation in the middle of the menstrual is fairly clear. Hormonal changes also occur in this period. Why the hormonal changes occur, and what their relationship is with the activation of the processes is unclear. That is also the case during pregnancies. What is it that determines that a pregnancy should last an average of 40 weeks? What causes the changes in a complicated pregnancy? What are those changes? Prostaglandin concentrations have been found to have some relationship with these changes, but the activation of these changes and how to examine them is unknown. Using an example from practical experience, this article illustrates what Horrobin and Manku already reported in 1977, namely, the properties of prostaglandin E1 and 6-keto pgF1α: reversal effect with elevated concentration. The properties described is exceptionally suitable for the time of activation in a biochemically regulated measure and regulation system. These properties can help explain the occurrence of physiological cycles. The known electronic saw-tooth wave has a biochemical analogue with this. This paper describes the presumed relationship between hormones and the accompanying prostaglandins with the hormone effects based on what is known regarding their concentrations progress. This relationship reveals the practical consequences of the experimentally found sensitivity of biochemical effects with regard to the accompanying prostaglandins. This paper shows how the theoretical relationship between effects of oestrogens and progestagens result in a curve that comprise observable aspects of the Basal Body Temperature Curve. The modulating and activating prostaglandins also affect local changes in blood circulation. These changes are visible on specific sites on the abdominal skin via viscerocutaneous reflex pathways. Changes in blood circulation at specific areas of the skin can be representative of pain. Pain that also frequently arises during activation processes. These changes can be seen and measured with non-contactual infrared thermography on the cutaneous surface, and moments of activation and pain can be determined.

Noise decomposition of intracellular biochemical signaling networks using nonequivalent reporters.

Experimental measurements of biochemical noise have primarily focused on sources of noise at the gene expression level due to limitations of existing noise decomposition techniques. Here, we introduce a mathematical framework that extends classical extrinsic-intrinsic noise analysis and enables mapping of noise within upstream signaling networks free of such restrictions. The framework applies to systems for which the responses of interest are linearly correlated on average, although the framework can be easily generalized to the nonlinear case. Interestingly, despite the high degree of complexity and nonlinearity of most mammalian signaling networks, three distinct tumor necrosis factor (TNF) signaling network branches displayed linearly correlated responses, in both wild-type and perturbed versions of the network, across multiple orders of magnitude of ligand concentration. Using the noise mapping analysis, we find that the c-Jun N-terminal kinase (JNK) pathway generates higher noise than the NF-κB pathway, whereas the activation of c-Jun adds a greater amount of noise than the activation of ATF-2. In addition, we find that the A20 protein can suppress noise in the activation of ATF-2 by separately inhibiting the TNF receptor complex and JNK pathway through a negative feedback mechanism. These results, easily scalable to larger and more complex networks, pave the way toward assessing how noise propagates through cellular signaling pathways and create a foundation on which we can further investigate the relationship between signaling system architecture and biological noise.

Assembly of adiponectin oligomers.

Adiponectin is among the most studied adipokines, the collection of molecules secreted from adipose tissue. It is also one of the most architecturally complex adipokines with its various oligomeric states that include trimers, hexamers, nonamers (9mers), dodecamers (12mers), and octadecamers (18mers). The importance of adiponectin in metabolic regulation is underscored by its strong positive associations with improvement in insulin action and also decreased risks for developing type 2 diabetes. Understanding the mechanisms involved in maintaining the steady-state concentrations of adiponectin oligomers in circulation is therefore likely to provide important insight into the development of insulin resistance. This review will discuss the current state of knowledge regarding the biochemical composition of adiponectin oligomers, the commonly used techniques to analyze them, and the known post-translational modifications that affect their assembly. Evidence based on in vitro oligomer assembly reactions in support of a "cystine ratchet" model of adiponectin oligomer formation will be considered along with limitations of the evidence. Secretory pathway proteins that have been shown to affect the distribution of adiponectin oligomers will also be discussed along with hypotheses regarding their potential involvement in the cystine ratchet model of adiponectin oligomerization.

Effect of sex and seasons of the year on hematologic and serum biochemical variables of captive brown brocket deer (Mazama gouazoubira) / Efeito do sexo e estações do ano sobre as variáveis hematológicas e bioquímico-séricas de veados-catingueiro (Mazama gouazoubira) criados em cativeiro

The Brown brocket deer (Mazama gouazoubira) is the most common free-living and captive deer in South America, especially in Brazil, and has great ecological and scientific significance. However, data on hematological and biochemical parameters in brown brocket deer are scarce. The goal of this study was to establish reference ranges for hematological and biochemical parameters of Mazama gouazoubira, comparing differences during the seasons of the year and between sex. Blood samples from ten adult healthy brown brocket deer (6 female and 4 male) were collected during daytime, monthly, during 12 months. The animals were maintained in individual stable, protected from noise and fed ad libitum with commercial ration and green fodder. For blood collection, animals were submitted to physical restrain for no longer than 2 minutes. The following parameters were determined: red blood cell count (RBC), haemoglobin concentration, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC), platelet count, enzyme activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) and serum levels of alkaline phosphatase (ALP), creatine kinase (CK), total protein (TP), albumin, cholesterol, total calcium, ionic calcium, sodium, potassium, magnesium, triglycerides, creatinine and urea. Values were compared according to season and sex. RBC count, WBC count and MCV suggested seasonal influence. Haemoglobin concentration, PCV and MCV were influenced by sex. Serum concentration of total calcium, ionic calcium, sodium, potassium and magnesium were influenced by season. Serum magnesium was also influenced by sex. The blood parameters herein reported may be useful as reference values for diagnostic and prognostic purposes in captive brown-brocket deer.

O veado-catingueiro (Mazama gouazoubira) é o cervídeo de vida livre mais comum na América do Sul, especialmente no Brasil, e tem grande importância ecológica e científica. No entanto, dados sobre parâmetros hematológicos e bioquímicos nesta espécie animal são escassos. O objetivo deste trabalho foi estabelecer valores de referência hematológica e bioquímica do Mazama gouazoubira, comparando diferenças entre as estações do ano e entre sexo. Amostras sanguíneas de dez veados-catingueiros (6 fêmeas e 4 machos), todos adultos hígidos, foram mensalmente colhidas pela manhã, durante 12 meses, e prontamente analisadas. Os animais foram mantidos em baias individual, protegidos de barulho e alimentados ad libitum com ração comercial e forragem verde. Para obtenção das amostras, os animais foram submetidos à contenção física por, no máximo, 2 minutos. Os seguintes parâmetros foram determinados: Contagem total de hemáceas, concentração de hemoglobina, volume globular (VG), volume corpuscular médio (VCM), hemoglobina corpuscular média (HCM), concentração de hemoglobina corpuscular média (CHCM), contagem total de leucócitos, contagem de plaquetas, atividade das enzimas alanina aminotransferase (ALT), aspartato aminotransferase (AST), gama glutamiltransferase (GGT) e concentrações séricas de fosfatase alcalina (FA), creatino quinase (CK), proteína total (PT), albumina, colesterol, cálcio total, cálcio iônico, sódio, potássio, magnésio, triglicérides, creatinina e uréia. Os valores foram comparados de acordo com o sexo e sazonalidade. A contagem total de hemáceas, de leucócitos e VCM sugeriu influência das estações do ano sobre estes parâmetros. A concentração de hemoglobina, VG e VCM foram influenciados pelo sexo. Concentrações séricas de cálcio total, cálcio iônico, sódio, potássio e magnésio também foram influenciados pelas estações do ano. O teor sérico de magnésio sofreu influência, ainda, do sexo. Os parâmetros sanguíneos obtidos e aqui reportados podem ser úteis como valores de referência nesta espécie para fins diagnóstico e prognóstico.

Complexity reduction preserving dynamical behavior of biochemical networks.

The complexity of biochemical systems, stemming from both the large number of components and the intricate interactions between these components, may hinder us in understanding the behavior of these systems. Therefore, effective methods are required to capture their key components and interactions. Here, we present a novel and efficient reduction method to simplify mathematical models of biochemical systems. Our method is based on the exploration of the so-called admissible region, that is the set of parameters for which the mathematical model yields some required output. From the shape of the admissible region, parameters that are really required in generating the output of the system can be identified and hence retained in the model, whereas the rest is removed. To describe the idea, first the admissible region of a very small artificial network with only three nodes and three parameters is determined. Despite its simplicity, this network reveals all the basic ingredients of our reduction method. The method is then applied to an epidermal growth factor receptor (EGFR) network model. It turns out that only about 34% of the network components are required to yield the correct response to the epidermal growth factor (EGF) that was measured in the experiments, whereas the rest could be considered as redundant for this purpose. Furthermore, it is shown that parameter sensitivity on its own is not a reliable tool for model reduction, because highly sensitive parameters are not always retained, whereas slightly sensitive parameters are not always removable.

Mass spectrometry-based proteomics approaches applied in cataract research.

Cataract, the opacification of the eye lens, is the leading cause of blindness worldwide--it accounts for approximately 42% of all cases. The lens fibers have the highest protein content within the body, more than 35% of their wet weight. Given the eye lens pure composition of highly abundant structural proteins crystallins (up to 90%), it seems to be an ideal proteomic entity to study and might be also hypothesized to model the other protein conformational diseases. Crystallins are extremely long-lived, and there is virtually no protein turnover. This provides great opportunities for post-translational modifications (PTM) to occur and to predispose lens to the cataract formation. Despite recent progress in proteomics, the human lens proteome remains largely unknown. Mass spectrometry hold great promise to determine which crystallin modifications lead to a cataract. Quantitative analysis of PTMs at the peptide level with proteomics is a powerful bioanalytical tool for lens-tissue samples, and provides more comprehensive results. New mass spectrometry-based approaches that are being applied to lens research will be highlighted. Finally, the future directions of proteomics cataract research will be outlined.

Pharmacological characterization of pannexin-1 currents expressed in mammalian cells.

Pannexin (Panx) 1 is a widely expressed protein that shares structural, but not amino acid, homology with gap junction proteins, the connexins. Panx1 does not form gap junctions in mammalian cells, but it may function as a plasma membrane hemichannel. Little is known of the pharmacological properties of panx1 expression in mammalian cells. Here, we identify three variants in the human PANX1 gene. We expressed these variants and mouse Panx1 in mammalian cells and compared Panx1-induced currents. All human Panx1 variants and the mouse Panx1 showed identical protein expression levels, localization patterns, and functional properties, although the frequency of functional expression was species-dependent. Panx1 currents were independent of changes in extracellular or intracellular calcium or phospholipase C transduction. We found compounds that inhibited Panx1 currents with a rank order of potency: carbenoxolone > disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) approximately disodium 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate approximately 5-nitro-2-(3-phenylpropylamino)benzoic acid > indanyloxyacetic acid 94 >> probenecid >> flufenamic acid = niflumic acid. Triphosphate nucleotides (ATP, GTP, and UTP) rapidly and reversibly inhibited Panx1 currents via mechanism(s) independent of purine receptors. When Panx1 was coexpressed with purinergic P2X(7) receptor (P2X(7)R), DIDS was found to act as a P2X(7)R antagonist to inhibit ATP-evoked currents, but none of the other compounds inhibited P2X(7)R currents. This is the first detailed pharmacological characterization of Panx1-mediated currents in mammalian cells and sheds new, although contradictory, light on the hypothesis that Panx1 acts as a hemichannel to allow passage of large molecules in response to P2X(7)R activation.

Biomechanical regulation of cell orientation and fate.

Biomechanical regulation of tumor phenotypes have been noted for several decades, yet the function of mechanics in the co-evolution of the tumor epithelium and altered cancer extracellular matrix has not been appreciated until fairly recently. In this review, we examine the dynamic interaction between the developing epithelia and the extracellular matrix, and discuss how similar interactions are exploited by the genetically modified epithelium during tumor progression. We emphasize the process of mechanoreciprocity, which is a phenomenon observed during epithelial transformation, in which tension generated within the extracellular microenvironment induce and cooperate with opposing reactive forces within transformed epithelium to drive tumor progression and metastasis. We highlight the importance of matrix remodeling, and present a new, emerging paradigm that underscores the importance of tissue morphology as a key regulator of epithelial cell invasion and metastasis.