Cold-Driven Hemoglobin Evolution in Antarctic Notothenioid Fishes Prior to Hemoglobin Gene Loss in White-Blooded Icefishes.

Expression of multiple hemoglobin isoforms with differing physiochemical properties likely helps species adapt to different environmental and physiological conditions. Antarctic notothenioid fishes inhabit the icy Southern Ocean and display fewer hemoglobin isoforms, each with less affinity for oxygen than temperate relatives. Reduced hemoglobin multiplicity was proposed to result from relaxed selective pressure in the cold, thermally stable, and highly oxygenated Antarctic waters. These conditions also permitted the survival and diversification of white-blooded icefishes, the only vertebrates living without hemoglobin. To understand hemoglobin evolution during adaptation to freezing water, we analyzed hemoglobin genes from 36 notothenioid genome assemblies. Results showed that adaptation to frigid conditions shaped hemoglobin gene evolution by episodic diversifying selection concomitant with cold adaptation and by pervasive evolution in Antarctic notothenioids compared to temperate relatives, likely a continuing adaptation to Antarctic conditions. Analysis of hemoglobin gene expression in adult hematopoietic organs in various temperate and Antarctic species further revealed a switch in hemoglobin gene expression underlying hemoglobin multiplicity reduction in Antarctic fish, leading to a single hemoglobin isoform in adult plunderfishes and dragonfishes, the sister groups to icefishes. The predicted high hemoglobin multiplicity in Antarctic fish embryos based on transcriptomic data, however, raises questions about the molecular bases and physiological implications of diverse hemoglobin isoforms in embryos compared to adults. This analysis supports the hypothesis that the last common icefish ancestor was vulnerable to detrimental mutations affecting the single ancestral expressed alpha- and beta-globin gene pair, potentially predisposing their subsequent loss.

A 3-alkylpyridine-bearing alkaloid exhibits potent antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) with no detectable resistance.

Staphylococcus aureus is a Gram-positive bacterium responsible for a wide variety of infectious diseases, and its methicillin-resistant isolates pose a serious worldwide public health risk. New drugs are urgently needed for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we evaluated the antibacterial activity of five 3-alkyl-pyridinic analogs against MRSA and, of these compounds, compound 6 showed promising antibacterial activity against Staphylococcus with minimum inhibitory concentration (MIC) ranging from 0.98 to 3.9 µgmL-¹ . In addition, it exhibited a rapid bactericidal action, with complete elimination of MRSA after 6 h of incubation at 15.6 µgmL-¹ . Compound 6 had the ability to damage the bacterial membrane and induce cell lysis and, due to its action on the membrane, showed low resistance induction potential in vitro. In the combination study, compound 6 revealed an additive effect (FICI = 1) with vancomycin and ofloxacin and ciprofloxacin (FICI = 0.75) against MRSA, reducing the effective concentration of this antibiotic two-fold. The anti-staphylococcal activity of compound 6 was stable in the presence of different concentrations of NaCl (50, 200, and 400 µM), trypsin ( 1:500, 1:250) and under a variety of pH values (4, 5, 6, and 8); however, its binding to plasmatic proteins (i.e., albumin) was substantial. The previous exposure of MRSA to the compound was able to reduce the formation of bacterial biofilm and reduce the biomass of mature biofilms. Compound 6 showed low selectivity in vitro for MRSA USA 300 when compared to eukaryotic cells (epithelial, fibroblast, and red blood cells).

Toxicological Effects of Roundup® on Drosophila melanogaster Reproduction.

Herbicide use has increased dramatically since 2001, particularly Roundup®. Effective in agricultural practice, Roundup® adversely affects non-target organisms, including reproductive and endocrine systems. We exposed fruit flies, Drosophila melanogaster, to either Roundup® Ready to Use, containing pelargonic acid and glyphosate, or Roundup® Super Concentrate, that includes glyphosate and POEA, at sublethal concentrations. Both Roundup® formulations reduced ovary volume with fewer mature oocytes, most adversely at the highest concentration tested. Flies exposed within 2 h of eclosion were affected more than at 4 h, suggesting a critical period of increased ovarian sensitivity. These results support multi-species evidence that glyphosate-based herbicides interfere with normal development of the reproductive systems of non-target organisms.

Antibacterial, antifungal, and antiviral activities of chalcone-bearing tetrahydropyranyl and 2,4-dihydroxyl moieties / Actividad antibacteriana, antifungica y antiviral de las chalconas que contienen los grupos tetrahidropiranilo y 2,4-dihidroxilo / Atividades antibacteriana, antifúngica e antiviral de chalconas funcionalizadas com os grupos tetrahidropiranil e 2,4-dihidroxil

SUMMARY Chalcones highlights as an important structure in medicinal chemistry and thus has been widely used as a template in the development of new drugs. In this study, we aim to determine the antibacterial, anti-Candida, and anti-Dengue potential of new chalcone-bearing 2,4-dihydroxyl and tetrahydropyranyl moieties. Antimicrobial activity assays showed that microorganism of the Staphylococcus genus (including methicillin-resistant strains) were susceptible to 2,4-dihydroxychalcones, with minimum inhibitory concentrations (MICs) ranging of 19.5 to 125 µg.mL-1. Compound 4e, which showed the highest bacteriostatic effect, also has bactericidal activity from of 80 µg.mL-1. The growth of oral isolates of Candida albicans was also efficiently inhibited with compound 4e (MIC: 15.6-32.3 µg.mL-1), which was fungicidal at 15.6 µg.mL-1. However, the presence of the tetrahydropyranyl moiety impaired both the antibacterial and antifungal effects. None of the chalcones tested were actives against Dengue virus serotype 2. In conclusion, the compound 4e showed good anti-Staphylococci and anti-Candida activity and may be a promising prototype for the development of new antimicrobial agents.

RESUMEN Las chalconas se destacan como una estructura importante en la química médica y, por lo tanto, se ha empleado como prototipo para el desarrollo de nuevos fármacos. En este estudio, nuestro objetivo fue determinar el potencial antibacteriano, anti Candida y anti-Dengue de las nuevas chalconas que poseen los grupos 2,4-dihidroxilo y tetrahidropiranilo. El ensayo de actividad antimicrobiana mostró que las bacterias del género Staphylococcus (incluidas las cepas resistentes a la meticilina) fueron sensibles a las 2,4-dihidroxicalconas estudiadas, con concentraciones inhibitorias mínimas (CIM) que oscilan entre 19,5 y 125 µg.mL-1. El compuesto 4e, que tuvo el mejor efecto bacteriostático, también mostró un efecto bactericida a partir de la concentración de 80 µg.mL-1. El crecimiento de los aislamientos orales de Candida albicans también se inhibió eficientemente con el compuesto 4e (CIM: 15.6-32.3 µg.mL-1), que fue fungicida a una concentración de 15.6 µg.mL-1. Sin embargo, la presencia del grupo tetrahidropiranilo perjudicó la actividad antibacteriana y anti-fúngica de los análogos de la chalcona. Además, ninguno de los compuestos evaluados mostró un efecto contra el virus del dengue serotipo 2. En conclusión, el compuesto 4e muestra una buena actividad anti-estafilocócica y anti-Candida y puede ser un prototipo prometedor para el desarrollo de nuevos agentes antimicrobianos.

RESUMO As chalconas se destacam como uma importante estrutura na química medicinal e dessa forma tem sido empregada como um protótipo para o desenvolvimento de novos fármacos. Nesse estudo, nós objetivamos determinar o potencial antibacteriano, anti-Candida, e anti-Dengue de novas chalconas que possuem os grupos 2,4-dihidroxil e tetrahidropiranil. O ensaio de atividade antimicrobiana mostrou que bactérias do gênero Staphylococcus (incluindo linhagens resistentes a meticilina) foram sensíveis para as 2,4-dihidroxichalconas estudadas, com concentrações inibitórias mínimas (CIM) variando de 19,5 para 125 Dengue potential of new chalcone µg.mL-1. O composto 4e, o qual apresentou o melhor efeito bacteriostático, também mostrou efeito bactericida a partir da concentração de 80 µg.mL-1. O crescimento de isolados orais de Candida albicans foi também eficientemente inibido com o composto 4e (CIM: 15.6-32.3 µg.mL-1), o qual foi fungicida a concentração de 15,6 µg.mL-1. Entretanto, a presença do grupo tetrahidropiranil prejudicou a atividade antibacteriana e antifúngica dos análogos de chalcona. Adicionalmente, nenhum dos compostos avaliados mostrou efeito contra o vírus da dengue sorotipo 2. Em conclusão, o composto 4e apresenta boa atividade anti-estafilocóccica e anti-Candida, e pode ser um promissor protótipo para o desenvolvimento de novos agentes antimicrobianos.

Changes in Proportions of Emergency Department Patients with Mental Illness Noted in the National Hospital Ambulatory Medical Care Survey, 2012-2015.

OBJECTIVES: The primary objective of this study was to determine whether there was a change in the rate and types of patients with psychiatric illnesses being seen in the emergency department (ED) from 2012 to 2015 using the National Ambulatory Care Survey. A secondary objective was to determine what if any changes occurred in the resources available to care for these patients. METHODS: Our study used 2012-2015 data from the National Hospital Ambulatory Medical Care Survey and the State Mental Health Agency Per Capita Mental Health Services Expenditures, and expenditures data from 2012-2015 to examine whether there was a significant change in the rate and type of mental illness ED visits. Additional data on the number of beds per region from the National Mental Health Services Survey, 2012-2015 were used. A t test was used to look for significant (P = 0.05) changes in the rate and types of patients, ED dispositions, ED reimbursement types, region and community level income, sex, age, state mental health funding, and psychiatric beds from 2012 to 2015. RESULTS: There was an 8% increase in the rate of patients who presented with a diagnosed mental health disorder (P = 0.03, 95% confidence interval [CI] 5.32-5.96) and substance use disorders (P = 0.03, 95% CI 0.564-0.122). The reimbursement for these visits did change (P = 0.01, 95% CI 0.245-0.685); however, there was no significant increase (P = 0.07, 95% CI-214 to 101) in state mental health budgets and the number of psychiatric and detox hospital beds from 2012 to 2015. CONCLUSIONS: The rate and types of mental health patients coming to the ED are still on the rise. This is coupled with a lack of mental health infrastructure to address the needs and diagnoses that continue to be seen in the ED. States may need increased, targeted funding for mental health outside the increase in coverage via the Patient Protection and Affordable Care Act to slow the rate of mental health patients seen in the ED.

First report of polymyxin B activity in Klebsiella pneumoniae biofilm.

Considering the clinical importance of biofilm in medical devices and chronic infections, this study aimed to investigate the action of polymyxin B on Klebsiella pneumoniae (K. pneumoniae) biofilm. The experiments were performed using a biofilm formation assay and the interaction of polysorbate 80 was explored. Both inhibition of biofilm formation and reduction of pre-formed biofilm occurred in a concentration-dependent manner with inhibition as high as 56 and 64%, and reduction of pre-formed biofilm as high as 70 and 66%, with and without polysorbate, respectively. The addition of polysorbate enhances the biofilm reduction, but more studies are needed to elucidate this mechanism. Our findings reveal, for the first time, polymyxin B as a potential agent for the treatment of K. pneumoniae biofilm, a current challenge for clinical practice.

l-Alanine activates hepatic AMP-activated protein kinase and modulates systemic glucose metabolism.

OBJECTIVE: AMP activated protein kinase (AMPK) is recognized as an important nutrient sensor contributing to regulation of cellular, tissue, and systemic metabolism. We aimed to identify specific amino acids which could modulate AMPK and determine effects on cellular and systemic metabolism. METHODS: We performed an unbiased amino acid screen to identify activators of AMPK. Detailed analysis of cellular signaling and metabolism was performed in cultured hepatoma cells, and in vivo glucose metabolism and metabolomic patterns were assessed in both chow-fed mice and mice made obese by high-fat diet feeding. RESULTS: Alanine acutely activates AMP kinase in both cultured hepatic cells and in liver from mice treated in vivo with Ala. Oral alanine administration improves systemic glucose tolerance in both chow and high fat diet fed mice, with reduced efficacy of Ala in mice with reduced AMPK activity. Our data indicate that Ala activation of AMPK is mediated by intracellular Ala metabolism, which reduces TCA cycle metabolites, increases AMP/ATP ratio, and activates NH3 generation. CONCLUSIONS: Ala may serve as a distinct amino acid energy sensor, providing a positive signal to activate the beneficial AMPK signaling pathway.

Inhibition of epithelial cell migration and Src/FAK signaling by SIRT3.

Metastasis remains the leading cause of cancer mortality, and reactive oxygen species (ROS) signaling promotes the metastatic cascade. However, the molecular pathways that control ROS signaling relevant to metastasis are little studied. Here, we identify SIRT3, a mitochondrial deacetylase, as a regulator of cell migration via its control of ROS signaling. We find that, although mitochondria are present at the leading edge of migrating cells, SIRT3 expression is down-regulated during migration, resulting in elevated ROS levels. This SIRT3-mediated control of ROS represses Src oxidation and attenuates focal adhesion kinase (FAK) activation. SIRT3 overexpression inhibits migration and metastasis in breast cancer cells. Finally, in human breast cancers, SIRT3 expression is inversely correlated with metastatic outcome and Src/FAK signaling. Our results reveal a role for SIRT3 in cell migration, with important implications for breast cancer progression.

Antibacterial activity of Lamiaceae plant extracts in clinical isolates of multidrug-resistant bacteria.

The antibacterial activity of plant extracts of the Lamiaceae family was evaluated against clinical isolates of multi-resistant Gram-negative bacteria by broth microdilution technique. Promising results were obtained considering that all extracts were active for at least two bacterial species with MIC ranging from 0.5 to 2.0 mg/mL.

Small-Molecule Screen Identifies De Novo Nucleotide Synthesis as a Vulnerability of Cells Lacking SIRT3.

Sirtuin 3 (SIRT3) is a NAD+-dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet (HFD)-induced metabolic disorders. Here, we performed a small-molecule screen and identified an unexpected metabolic vulnerability associated with SIRT3 loss. Azaserine, a glutamine analog, was the top compound that inhibited growth and proliferation of cells lacking SIRT3. Using stable isotope tracing of glutamine, we observed its increased incorporation into de novo nucleotide synthesis in SIRT3 knockout (KO) cells. Furthermore, we found that SIRT3 KO cells upregulated the diversion of glutamine into de novo nucleotide synthesis through hyperactive mTORC1 signaling. Overexpression of SIRT3 suppressed mTORC1 and growth in vivo in a xenograft tumor model of breast cancer. Thus, we have uncovered a metabolic vulnerability of cells with SIRT3 loss by using an unbiased small-molecule screen.

Effects of the 2009 Medical Cannabinoid Legalization Policy on Hospital Use for Cannabinoid Dependency and Persistent Vomiting.

BACKGROUND & AIMS: In 2009, the U.S. Department of Justice issued a memo stating that it would not prosecute users and sellers who complied with the state laws allowing for medical use of marijuana. There are growing concerns about legalization of marijuana use and its related public health effects. We performed an interrupted time series analysis to evaluate these effects. METHODS: We collected a representative sample of hospital discharge data from the Healthcare Cost and Utilization Project, from January 1993 to December 2014. We divided the data in to 3 groups: the prelegalization period (1993-2008), the legalization period (2009), and the postlegalization period (2010-2014). The disease variables were International Classification of Disease-Ninth Revision-Clinical Modification 304.30 cannabinoid dependency unspecified (CDU), 536.2 persistent vomiting, and an aggregate of CDU and persistent vomiting. We performed interrupted time series and Poisson-Gamma regression analysis to calculate each year's incidence rate of unspecified and persistent vomiting and CDU per 100,000 hospital discharges. CDU, persistent vomiting, and aggregate of CDU and persistent vomiting were modeled separately to estimate average incidence rate ratio and 95% confidence interval for each study phase. RESULTS: We observed an increasing trend of CDU or an aggregate of CDU and persistent vomiting during the prelegalization period. The legalization of marijuana significantly increased the incidence rate during the legalization period (by 17.9%) and the yearly average increase in rate by 6% after policy implementation, compared to the prelegalization period. The increase in rate of persistent vomiting after policy implementation increased significantly (by about 8%), although there were no significant trends in increase prior to or during marijuana legalization in 2009. CONCLUSIONS: In an interrupted time series analysis of before, during, and after medical marijuana legalization, we estimated levels and rate changes in CDU and persistent vomiting. We found persistent increases in rates of CDU and persistent vomiting during and after legalization of marijuana.

Intersections between mitochondrial sirtuin signaling and tumor cell metabolism.

Cancer cells use glucose and glutamine to facilitate cell growth and proliferation, a process coined "metabolic reprograming" - an emerging hallmark of cancer. Inside the cell, these nutrients synergize to produce metabolic building blocks, such as nucleic acids, lipids and proteins, as well as energy (ATP), glutathione and reducing equivalents (NADPH), required for survival, growth and proliferation. Intense research aimed at understanding the underlying cause of the metabolic rewiring has revealed that established oncogenes and tumor suppressors involved in signaling alter cellular metabolism to contribute to the transition from a normal quiescent cell to a rapidly proliferating cancer cell. Likewise, bona fide metabolic sensors are emerging as regulators of tumorigenesis. This review will focus on one such family of sensors, sirtuins, which utilize NAD(+) as a cofactor to catalyze deacetylation, deacylation and ADP-ribosylation of their protein substrates. In this review, we will enumerate how cancer cell metabolism is different from a normal quiescent cell and highlight the emerging role of mitochondrial sirtuin signaling in the regulation of tumor metabolism.

PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis.

Cancer cells can divert metabolites into anabolic pathways to support their rapid proliferation and to accumulate the cellular building blocks required for tumour growth. However, the specific bioenergetic profile of invasive and metastatic cancer cells is unknown. Here we report that migratory/invasive cancer cells specifically favour mitochondrial respiration and increased ATP production. Invasive cancer cells use the transcription coactivator peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A, also known as PGC-1α) to enhance oxidative phosphorylation, mitochondrial biogenesis and the oxygen consumption rate. Clinical analysis of human invasive breast cancers revealed a strong correlation between PGC-1α expression in invasive cancer cells and the formation of distant metastases. Silencing of PGC-1α in cancer cells suspended their invasive potential and attenuated metastasis without affecting proliferation, primary tumour growth or the epithelial-to-mesenchymal program. Inherent genetics of cancer cells can determine the transcriptome framework associated with invasion and metastasis, and mitochondrial biogenesis and respiration induced by PGC-1α are also essential for functional motility of cancer cells and metastasis.

Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells.

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation.

Expression of the six chromate ion transporter homologues of Burkholderia xenovorans LB400.

The chromate ion transporter (CHR) superfamily comprises transporters that confer chromate resistance by extruding toxic chromate ions from cytoplasm. Burkholderia xenovorans strain LB400 has been reported to encode six CHR homologues in its multireplicon genome. We found that strain LB400 displays chromate-inducible resistance to chromate. Susceptibility tests of Escherichia coli strains transformed with cloned B. xenovorans chr genes indicated that the six genes confer chromate resistance, although under different growth conditions, and suggested that expression of chr genes is regulated by sulfate. Expression of chr genes was measured by quantitative reverse transcription-PCR (RT-qPCR) from total RNA of B. xenovorans LB400 grown under different concentrations of sulfate and exposed or not to chromate. The chr homologues displayed distinct expression levels, but showed no significant differences in transcription under the various sulfate concentrations tested, indicating that sulfate does not regulate chr gene expression in B. xenovorans. The chrA2 gene, encoded in the megaplasmid, was the only chr gene whose expression was induced by chromate and it was shown to constitute the chromate-responsive chrBACF operon. These data suggest that this determinant is mainly responsible for the B. xenovorans LB400 chromate resistance phenotype.

Clasificación e identificación de especies de actinomicetos: Un estudio fenotípico comparativo / Classification and identification of Actinomyces species: A comparative phenotype study

Las pruebas fisiológicas y bioquímicas constituyen dos de las principales metodologías utilizadas, principalmente en los laboratorios de microbiología, para la identificación y diferenciación de los actinomicetos. La finalidad de este trabajo fue comparar y evaluar los métodos fenotípicos que son utilizados de manera rutinaria en la identificación de estos microorganismos. Se estudiaron setenta y tres cepas de actinomicetos provenientes de tres laboratorios de microbiología de Venezuela. El comportamiento fisiológico y bioquímico de las cepas en estudio fue evaluado mediante pruebas de descomposición de diferentes sustratos. Los resultados obtenidos permitieron observar diferencias en la identificación preliminar de las cepas realizada por estos laboratorios, conduciendo algunas veces a una nueva identificación de las mismas, gracias a la utilización de estos sustratos. Se pudo observar que la metodología empleada permitió la reclasificación taxonómica de casi todas las cepas estudiadas, lo que sugiere que es necesario estandarizar la metodología de identificación para los actinomicetos.

Physiologic and biochemical tests constitute two of the main methodologies, mainly used in microbiology laboratories, for the identification and differentiation of actinomyces. The purpose of this work was to compare and evaluate the phenotypic methods used routinely for the identification of these microorganisms. The study included seventy three actinomyces strains from three microbiology laboratories in Venezuela. The physiologic and biochemical behavior of the strains was evaluated through decomposition tests using different substrates. The results obtained showed differences in the preliminary identification of the strains done in the various laboratories, leading occasionally to a new identification arisen from the use of these substrates. It was seen that with the methods used it was possible to taxonomically reclassify almost all the strains studied, suggesting that it is necessary to standardize the methods for the identification of actinomyces.

Primer reporte de infección natural por Nocardia cyriacigeorgica en ratones BALB/c / First report of a natural infection by Nocardia cyriacigeorgica in BALB/c mice

Las bacterias del género Nocardia son actinomicetos aerobios cuyo hábitat es el suelo y la materia orgánica en descomposición. La infección natural por especies de este género ha sido reportada en humanos y animales, sin embargo, la infección natural en ratones no ha sido descrita. En este estudio se demuestra por primera vez el aislamiento de la especie recientemente descrita, Nocardia cyriacigeorgica, en pulmones de ratones Balb/c. El estudio microbiológico de las muestras de pulmón reportó la presencia de filamentos grampositivos ramificados fragmentados en forma cocobacilares. Los estudios de PCR demostraron que la especie aislada fue N. cyriacigeorgica. Se evidencia en este trabajo que N. cyriacigeorgica es potencialmente patógena en ratones Balb/c.

Bacteria belonging to the Nocardia genus are aerobic actinomycetae whose habitat is the ground and decomposing organic material. Natural infection by species belonging to this genus has been reported in humans and animals; nevertheless, natural infection in mice has not been described until now. This study demonstrates for the first time the isolation of a recently described species, Nocardia cyriacigeorgica, rom the lungs of Balb/c mice. The microbiological study of the lung samples reported the presence of branched Gram positive filaments with a cocobacillar shape. PCR studies demonstrated that the species isolated was N. cyriacigeorgica. This study demonstrates that N. cyriacigeorgica is potentially pathogenic for Balb/c mice.