Isolation and characterization of an atypical Mycoplasma gallisepticum strain showing a new mgc2 variant.

Mycoplasma gallisepticum (MG) is an important pathogen of the poultry industry able to cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite the application of biosecurity measures and the availability of vaccines for chickens, monitoring systems routinely applied for MG detection are still essential for infection control. Pathogen isolation is time-consuming and not suitable for rapid detection, albeit it is a compulsory step for genetic typing and antimicrobial susceptibility evaluation of single strains. The mgc2 gene is a species-specific molecular target adopted by most of the PCR protocols available for MG diagnosis, which are also included in the WOAH Terrestrial Manual. We describe the case of an atypical MG strain, isolated in 2019 from Italian turkeys, characterized by an mgc2 sequence not detectable by common endpoint PCR primers. Considering the potential risk of false negative results during diagnostic screenings with the endpoint protocol, the authors propose an alternative mgc2 PCR endpoint protocol, named MG600, which should be considered as a further diagnostic tool.

Processed meat consumption and risk of cancer: a multisite case-control study in Uruguay.

BACKGROUND: The role of processed meat in the aetiology of several cancers was explored in detail. METHODS: In the time period 1996-2004, a multisite case-control study was conducted in Montevideo, Uruguay. The study included 6 060 participants (3 528 cases and 2 532 controls) corresponding to cancers of the oral cavity, pharynx, oesophagus, stomach, colon, rectum, larynx, lung, female breast, prostate, urinary bladder, and kidney (renal cell carcinoma only). RESULTS: The highest odds ratios (ORs) were positively associated with cancers of the colon, rectum, stomach, oesophagus, and lung. With the exception of renal cell carcinoma, the remaining cancer sites were significantly associated with elevated risks for processed meat consumption. Furthermore, mortadella, salami, hot dog, ham, and salted meat were strongly associated with risk of several cancer sites. CONCLUSION: It could be concluded that processed meat intake could be a powerful multiorgan carcinogen.

Dietary folate intake and the risk of 11 types of cancer: a case-control study in Uruguay.

BACKGROUND: There is limited, but inconclusive, epidemiological evidence that high folate intake decreases the risk of colorectal and esophageal cancers. For other cancer sites, the evidence is even less consistent or extensive. MATERIALS AND METHODS: We conducted a case-control study of dietary folate intake and risk of 11 cancer sites in Uruguay between 1996 and 2004, including 3539 cancer cases and 2032 hospital controls. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of cancer associated with folate intake. RESULTS: In the multivariable model, there was a significant decrease in the risk of cancers of the oral cavity and pharynx (OR = 0.49, 95% CI 0.24-0.98), esophagus (OR = 0.29, 95% CI 0.14-0.60), upper aerodigestive tract (OR = 0.41, 95% CI 0.26-0.65), colorectum (OR = 0.42, 95% CI 0.23-0.76) and kidney (OR = 0.35, 95% CI 0.13-0.93) for the highest versus the lowest quartile of dietary folate intake. CONCLUSIONS: Our results not only confirm earlier findings of decreased risk of colorectal and esophageal cancers with a high dietary folate intake but also suggest decreased risk of several other cancers. However, we cannot exclude the possibility that residual confounding, multiple comparisons or other forms of bias could explain these results.

The safety profile of probiotic VSL#3®. A meta-analysis of safety data from double-blind, randomized, placebo-controlled clinical trials.

The article "The safety profile of probiotic VSL#3®. A meta-analysis of safety data from double-blind, randomized, placebo-controlled clinical trials", by V. Panetta, A. Bacchieri, S. Papetti, E. De Stefani, P. Navarra, published in Eur Rev Med Pharmacol Sci 2020; 24 (2): 963-973-DOI: 10.26355/eurrev_202001_20082-PMID: 32017005, has been retracted based on commentary received from a new set of reviewers.  The authors will be able to resubmit a new article addressing the reviewers' comments for the Journal's consideration. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20082.

The safety profile of probiotic VSL#3®. A meta-analysis of safety data from double-blind, randomized, placebo-controlled clinical trials.

OBJECTIVE: A high-concentration of a multi-strain probiotic mixture, VSL#3® is widely used 'whenever it is useful to promote the balance of intestinal flora'. As a food supplement, VSL#3® has been so far scarcely investigated on the aspect of safety. To fill this gap, in this paper, we analyzed the adverse events (AEs) recorded during the conduct of three (3) double-blind, randomized, placebo-controlled trials carried out to explore the efficacy of VSL#3® in various clinical settings. Data from a large open-label observational trial were also considered. MATERIALS AND METHODS: All trials included in the analysis were carried out according to good clinical practice (GCP) rules. AEs were classified by System Organ Class (SOC), Preferred Term (PT) and frequency. Differences vs. placebo control were considered as statistically significant if the p-value was < 0.05. RESULTS: A total of 120 patients were analyzed, 70 patients being included in the randomized controlled trials. In this population, 45 patients had at least one AE, 20 (64.5%) in the placebo group and 25 (64.1%) in the VSL#3® group. 29 patients had at least one related AE, 14 (45.2%) and 15 (38.5%) in the two treatment groups, respectively. Only one AE was assessed as serious, i.e., Foetal malformation, which occurred in the placebo group and was considered unrelated. No significant difference was found between VSL#3® and placebo for any of the SOC considered, with the exception of Injury, poisoning and procedural complications, which was in favor of VSL#3®. CONCLUSIONS: Based on GCP-quality data from clinical trials, we conclude that VSL#3® is a safe and well-tolerated agent.

Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.

Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming alpha-subunits (MaxiKalpha) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiKalpha is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiKalpha was visualized by three independent labeling approaches: (1) MaxiKalpha-specific antibodies, (2) expressed EGFP-labeled MaxiKalpha, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiKalpha association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiKalpha labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+-release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKalpha surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a approximately 3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK stores to the plasma membrane of developing murine astrocytes.

Ionic mechanisms of cholinergic excitation in molluscan neurons.

Acetylcholine appears to be an excitatory transmitter at synapses on two different types of molluscan nerve cells: the so-called D- and CILDA neurons. The action of this substance is different in the two cases. In D-neurons, this compound increases the permeability of the subsynaptic or somatic membrane to chloride ions, and through a net efflux of this anion, depolarizes the cell. In CILDA neurons, on the other hand, acetylcholine depolarzies the cell by increasing its permeability to sodium ions.

Gating current noise produced by elementary transitions in Shaker potassium channels.

Gating currents provide a direct record of the spatial rearrangement of charges occurring within the protein of voltage-sensitive ion channels. If the elementary charges move as very brief discrete pulses of current, they will produce fluctuations in the macroscopic gating current. The variance of such fluctuations in gating currents was measured in Shaker potassium channels expressed in Xenopus oocytes with a sufficiently high recording bandwidth to estimate the magnitude and time distribution of the elementary transition charge movements. Channel activation occurred in two sequential stages. The first stage consisted of numerous, fast transitions, each moving small amounts of charge that contributed little to the fluctuation in gating current, whereas the second stage, which contributed the bulk of the fluctuation, was represented by a number of discrete, correlated transitions, one or more of which carried a charge of at least 2.4 elementary charges across the membrane field.

Potentiation by the beta subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel.

The voltage-activated rabbit cardiac calcium channel alpha 1 subunit was expressed in Xenopus oocytes. The charge movement of its voltage sensor was measured and related to the opening of the ion-conducting pore. The half-activation potential for charge movement was 35 millivolts more negative than that for pore opening. Coexpression of the cardiac calcium channel beta subunit reduced this difference without affecting charge movement. Thus, intramolecular coupling between the voltage sensor and the channel pore opening can be facilitated by a regulatory subunit.

Molecular basis of gating charge immobilization in Shaker potassium channels.

Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.

Voltage-dependent proton transport by the voltage sensor of the Shaker K+ channel.

In voltage-dependent ion channels, pore opening is initiated by electrically driven movements of charged residues, and this movement generates a gating current. To examine structural rearrangements in the Shaker K+ channel, basic residues R365 and R368 in the S4 segment were replaced with histidine, and gating currents were recorded. Changes in gating charge displacement with solvent pH reveal voltage-dependent changes in exposure of the histidine to solvent protons. This technique directly monitors accessibility changes during gating, probes the environment even in confined locations, and introduces minimal interference of gating charge motion. The results indicate that charges 365 and 368 traverse the entire electric field during gating. The remarkable implication of the successive exposure of histidine to each side of the membrane is that in a pH gradient, the voltage sensor transports protons.

Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels.

In voltage-dependent ion channels, a voltage sensor region is responsible for channel activation and an aqueous pore is responsible for ion conduction. These two processes have been traditionally considered to be independent. We describe here a mutation in the putative pore region (W434F) that completely abolishes ion conduction without affecting the gating charge of the channel. Gating currents in the nonconductive mutant were found to be identical in their kinetic and steady-state properties to those in conductive channels. Gating current measurements could be performed without subtracting pulses and in the presence of normal physiological solutions. Application of internal tetraethylammonium (an open channel blocker) induced Off charge immobilization for large depolarizations, suggesting that the internal tetraethylammonium-binding site becomes available upon depolarization. We concluded that for this mutant, although the conduction pathway is not functional, the channel can still undergo the closed-open conformation in response to voltage changes.

The amino terminus of a calcium channel beta subunit sets rates of channel inactivation independently of the subunit's effect on activation.

There is molecular diversity in both alpha 1 and beta subunits of voltage-gated Ca2+ channels. Coupling between voltage sensing and pore opening of the C-type alpha 1 (alpha 1c) is improved by the type 2 beta subunit (beta 2), and E-type alpha 1 beta complexes inactivate at different rates depending on the nature of beta. We compared the effects of type 1 and 2 beta subunits on activation of the human E-type alpha 1 (alpha 1E) with the effects they have on inactivation, as seen in Xenopus oocytes. The beta subtypes stimulated activation in similar fashion but affected inactivation differently, and even in opposing directions. beta subunits have a common central core but differ in their N- and C-termini and in a central region. N-terminal chimeras between beta 1 and beta 2 subunits that have opposing effects on inactivation resulted in the reciprocal transfer of their effects. We conclude that regulation of activation and inactivation of alpha 1 by beta are separable events and that the N-terminus of beta is one of the structural determinants important in setting the rate and voltage at which an alpha 1 inactivates.

Endocytic trafficking signals in KCNMB2 regulate surface expression of a large conductance voltage and Ca(2+)-activated K+ channel.

Large conductance voltage and calcium-activated K(+) channels play critical roles in neuronal excitability and vascular tone. Previously, we showed that coexpression of the transmembrane beta2 subunit, KCNMB2, with the human pore-forming alpha subunit of the large conductance voltage and Ca(2+)-activated K(+) channel (hSlo) yields inactivating currents similar to those observed in hippocampal neurons [Hicks GA, Marrion NV (1998) Ca(2+)-dependent inactivation of large conductance Ca(2+)-activated K(+) (BK) channels in rat hippocampal neurones produced by pore block from an associated particle. J Physiol (Lond) 508 (Pt 3):721-734; Wallner M, Meera P, Toro L (1999b) Molecular basis of fast inactivation in voltage and Ca(2+)-activated K(+) channels: A transmembrane beta-subunit homolog. Proc Natl Acad Sci U S A 96:4137-4142]. Herein, we report that coexpression of beta2 subunit with hSlo can also modulate hSlo surface expression levels in HEK293T cells. We found that, when expressed alone, beta2 subunit appears to reach the plasma membrane but also displays a distinct intracellular punctuated pattern that resembles endosomal compartments. beta2 Subunit coexpression with hSlo causes two biological effects: i) a shift of hSlo's intracellular expression pattern from a relatively diffuse to a distinct punctated cytoplasmic distribution overlapping beta2 expression; and ii) a decrease of hSlo surface expression that surpassed an observed small decrease in total hSlo expression levels. beta2 Site-directed mutagenesis studies revealed two putative endocytic signals at the C-terminus of beta2 that can control expression levels of hSlo. In contrast, a beta2 N-terminal consensus endocytic signal had no effect on hSlo expression levels. Thus, beta2 subunit not only can influence hSlo currents but also has the ability to limit hSlo surface expression levels via an endocytic mechanism. This new mode of beta2 modulation of hSlo may depend on particular coregulatory mechanisms in different cell types.

Hot infusions and risk of colorectal cancer in Uruguay: a case-control study.

BACKGROUND/OBJECTIVES: The evidence of possible roles for the most common hot infusions intake (tea and coffee) in the risk of colorectal cancer (CRC) needs additional data. Regarding 'mate' intake (infusion of Ilex paraguariensis herb), a previous multi-site study reported lack of association for its highest intake on CRC risk. The present study was conducted to better understand the associations between the intake of this and other infusions and CRC risk. SUBJECTS/METHODS: Patients (611 CRC incident cases and 2394 controls, all belonging to public hospitals) were interviewed through a questionnaire, including socio-demographic, reproductive and lifestyle variables, and a food-frequency questionnaire of 64 items, analyzing tea, 'mate' and coffee intake (consumer status, daily intake, age at start and at quit). Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated through unconditional logistic regression, adjusting for relevant potential confounders. RESULTS: Tea and coffee intake displayed significant and inverse associations with CRC risk, mainly among men (OR=0.54, 95% CI 0.38-0.76 for tea and OR=0.59, 95% CI 0.41-0.85 for coffee). Mate intake showed a significant inverse association among women (OR=0.50, 95% CI 0.33-0.77), with a marginal heterogeneity between sexes (P=0.07). Concerning age strata, tea intake displayed inverse associations in all ages, whereas 'mate' and coffee intake showed stronger inverse associations for age ⩾70, suggesting a gradient along time. CONCLUSIONS: We found evidence of different significant inverse associations for tea, 'mate' and coffee intake and CRC risk. To our knowledge, this is the first epidemiologic study reporting inverse results on 'mate' intake and CRC, which are explained by a stronger association among women.European Journal of Clinical Nutrition advance online publication, 23 August 2017; doi:10.1038/ejcn.2017.130.

Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities.

U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. Here, we report structures of the U6 RNA processing enzyme Usb1 from yeast and a substrate analog bound complex from humans. Unlike the human ortholog, we show that yeast Usb1 has cyclic phosphodiesterase activity that leaves a terminal 3' phosphate which prevents overprocessing. Usb1 processing of U6 RNA dramatically alters its affinity for cognate RNA-binding proteins. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp1, Prp24, Usb1 and Lsm2-8) and anti-cooperative interactions between Prp24 and Lhp1. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome.The mechanism of U6 small nuclear ribonucleoprotein (snRNP) biogenesis is not well understood. Here the authors characterize the enzymatic activities and structures of yeast and human U6 RNA processing enzyme Usb1, reconstitute post-transcriptional assembly of yeast U6 snRNP in vitro, and propose a model for U6 snRNP assembly.

KCNMB1 regulates surface expression of a voltage and Ca2+-activated K+ channel via endocytic trafficking signals.

Voltage-dependent and calcium-activated K(+) (MaxiK, BK) channels are ubiquitously expressed and have various physiological roles including regulation of neurotransmitter release and smooth muscle tone. Coexpression of the pore-forming alpha (hSlo) subunit of MaxiK channels with a regulatory beta1 subunit (KCNMB1) produces noninactivating currents that are distinguished by high voltage/Ca(2+) sensitivities and altered pharmacology [McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, Leonard RJ (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14:645-650; Wallner M, Meera P, Ottolia M, Kaczorowski G, Latorre R, Garcia ML, Stefani E, Toro L (1995) Characterization of and modulation by a beta-subunit of a human maxi K(Ca) channel cloned from myometrium. Receptors Channels 3:185-199]. We now show that beta1 can regulate hSlo traffic as well, resulting in decreased hSlo surface expression. beta1 subunit expressed alone is able to reach the plasma membrane; in addition, it exhibits a distinct intracellular punctated pattern that colocalizes with an endosomal marker. Coexpressing beta1 subunit with hSlo, switches hSlo's rather diffuse intracellular expression to a punctate cytoplasmic localization that overlaps beta1 expression. Furthermore, coexpressed beta1 subunit reduces steady-state hSlo surface expression. Site-directed mutagenesis underscores a role of a putative endocytic signal at the beta1 C-terminus in the control of hSlo surface expression. We propose that aside from its well-established role as regulator of hSlo electrical activity, beta1 can regulate hSlo expression levels by means of an endocytic mechanism. This highlights a new beta1 subunit feature that regulates hSlo channels by a trafficking mechanism.

Decreased expression of voltage- and Ca(2+)-activated K(+) channels in coronary smooth muscle during aging.

Aging is the main risk factor for coronary artery disease. One characteristic of aging coronary arteries is their enhanced contractile responses to endothelial vasoconstricting factors, which increase the risk of coronary vasospasm in older people. Because large-conductance voltage- and Ca(2+)-activated K(+) channels (MaxiK) are key regulators of vascular tone, we explored the possibility that this class of channels is diminished with increasing age. Using site-directed antibodies recognizing the pore-forming alpha subunit and electrophysiological methods, we demonstrate that the number of MaxiK channels is dramatically diminished in aged coronary arteries from old F344 rats. Channel density was reduced from 52+/-9 channels/pF (3 months old) to 18+/-5 channels/pF (25 to 30 months old), which represents a 65% reduction in the older population. Pixel intensity of Western blots was also diminished by approximately 50%. Moreover, the age-related decrease in the channel protein expression was also evident in humans, which showed approximately 80% reduction in 61- to 70-year-old subjects compared with 3- to 18-year-old youngsters and approximately 45% reduction compared with 19- to 56-year-old adults. In agreement with a reduction of MaxiK channel numbers in aging coronary arteries, old coronary arteries from F344 rats contract less effectively ( approximately 70% reduction) than young coronary arteries when exposed to the MaxiK channel blocker iberiotoxin. The contraction studies indicate that under physiological conditions, MaxiK channels are tonically active, serving as a hyperpolarizing force that opposes contraction. Thus, reduced expression of MaxiK channels in aged coronary arteries would lead to a decreased vasodilating capacity and increased risk of coronary spasm and myocardial ischemia in older people.

Esophageal cancer in Uruguay: a case-control study.

Esophageal cancer has constituted a major public health problem in Uruguay, with age-adjusted death rates of 14.5 X 10(5) for males and of 3.8 X 10(5) for females. A case-control study was undertaken to ascertain the possible association of the local custom of drinking infusions of Ilex paraguariensis ("maté") with cancer of the esophagus, after controlling for well-known risk factors, such as alcohol and tobacco consumption. Two hundred twenty-six patients with esophageal cancer and 469 controls (control:case = 2.1) were interviewed at the time of admission or consultation at the Oncology Institute of Montevideo from 1979 through 1984. Males showed elevated risks of esophageal cancer associated with heavy tobacco [relative risk (RR) = 10.8] and alcohol (RR = 10.3) exposures. Among females, the independent effects of tobacco and alcohol were nonsignificant. Maté consumption had an independent effect in both males and females, with odds ratios of 6.5 and 34.6, respectively, for heavy users. Moreover, a well-defined dose response was evident in both sexes.