Growth Phase-Dependent Chromosome Condensation and Heat-Stable Nucleoid-Structuring Protein Redistribution in Escherichia coli under Osmotic Stress.

The heat-stable nucleoid-structuring (H-NS) protein is a global transcriptional regulator implicated in coordinating the expression of over 200 genes in Escherichia coli, including many involved in adaptation to osmotic stress. We have applied superresolved microscopy to quantify the intracellular and spatial reorganization of H-NS in response to a rapid osmotic shift. We found that H-NS showed growth phase-dependent relocalization in response to hyperosmotic shock. In stationary phase, H-NS detached from a tightly compacted bacterial chromosome and was excluded from the nucleoid volume over an extended period of time. This behavior was absent during rapid growth but was induced by exposing the osmotically stressed culture to a DNA gyrase inhibitor, coumermycin. This chromosomal compaction/H-NS exclusion phenomenon occurred in the presence of either potassium or sodium ions and was independent of the presence of stress-responsive sigma factor σS and of the H-NS paralog StpA.IMPORTANCE The heat-stable nucleoid-structuring (H-NS) protein coordinates the expression of over 200 genes in E. coli, with a large number involved in both bacterial virulence and drug resistance. We report on the novel observation of a dynamic compaction of the bacterial chromosome in response to exposure to high levels of salt. This stress response results in the detachment of H-NS proteins and their subsequent expulsion to the periphery of the cells. We found that this behavior is related to mechanical properties of the bacterial chromosome, in particular, to how tightly twisted and coiled is the chromosomal DNA. This behavior might act as a biomechanical response to stress that coordinates the expression of genes involved in adapting bacteria to a salty environment.

Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance.

UNLABELLED: Potassium (K(+)) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K(+) and a variety of K(+) transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K(+) acquisition in Streptococcus mutans and the importance of K(+) homeostasis for its virulence attributes. The S. mutans genome harbors four putative K(+) transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K(+) cotransporter (GlnQHMP), and a channel-like K(+) transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K(+)] less than 5 mM eliminated biofilm formation in S. mutans. The functionality of the Trk2 system was confirmed by complementing an Escherichia coli TK2420 mutant strain, which resulted in significant K(+) accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K(+)-dependent cellular response of S. mutans to environment stresses. IMPORTANCE: Biofilm formation and stress tolerance are important virulence properties of caries-causing Streptococcus mutans. To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment of S. mutans. K(+) is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K(+) transporters in S. mutans. We identified the most important system for K(+) homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K(+) for the activity of biofilm-forming enzymes, which explains why such high levels of K(+) would favor biofilm formation.

Regulation of the competence pathway as a novel role associated with a streptococcal bacteriocin.

The oral biofilm organism Streptococcus mutans must face numerous environmental stresses to survive in its natural habitat. Under specific stresses, S. mutans expresses the competence-stimulating peptide (CSP) pheromone known to induce autolysis and facilitate the uptake and incorporation of exogenous DNA, a process called DNA transformation. We have previously demonstrated that the CSP-induced CipB bacteriocin (mutacin V) is a major factor involved in both cellular processes. Our objective in this work was to characterize the role of CipB bacteriocin during DNA transformation. Although other bacteriocin mutants were impaired in their ability to acquire DNA under CSP-induced conditions, the ΔcipB mutant was the only mutant showing a sharp decrease in transformation efficiency. The autolysis function of CipB bacteriocin does not participate in the DNA transformation process, as factors released via lysis of a subpopulation of cells did not contribute to the development of genetic competence in the surviving population. Moreover, CipB does not seem to participate in membrane depolarization to assist passage of DNA. Microarray-based expression profiling showed that under CSP-induced conditions, CipB regulated ∼130 genes, among which are the comDE locus and comR and comX genes, encoding critical factors that influence competency development in S. mutans. We also discovered that the CipI protein conferring immunity to CipB-induced autolysis also prevented the transcriptional regulatory activity of CipB. Our data suggest that besides its role in cell lysis, the S. mutans CipB bacteriocin also functions as a peptide regulator for the transcriptional control of the competence regulon.

Prevalence of malnutrition-inflammation complex syndrome and its correlation with thyroid hormones in chronic haemodialysis patients. / Prevalencia del síndrome complejo de malnutrición e inflamación y su correlación con las hormonas tiroideas en pacientes en hemodiálisis crónica.

INTRODUCTION: Low levels of thyroid hormones, total triiodothyronine (T3) and free triiodothyronine (FT3) in haemodialysis patients is a marker of malnutrition and inflammation and are predictors of mortality. The aim of this study was to determine the prevalence of malnutrition-inflammation complex syndrome in haemodialysis and its relationship with the thyroid hormones thyrotropin, T3, FT3 and free thyroxine (FT4), as well as to evaluate the prevalence of low FT3 syndrome and its correlation with nutritional and inflammatory markers. MATERIALS AND METHODS: Cross-sectional, analytical and comparative study that enrolled 128 haemodialysis patients: 50.8% females; mean age 45.05±17.01 years; mean time on haemodialysis 45.4±38.8 months; 29.7% diabetics; 79.7% with hypertension. Serum thyroid hormones thyrotropin, T3, FT3 and FT4 concentrations were measured and Malnutritition-Inflammation Score (MIS) was applie to diagnostic. RESULTS: Mean thyroid hormone values were: thyroid hormones thyrotropin 2.48±1.8 mIU/ml (range: 0.015-9.5), T3 1.18±0.39 ng/ml (range 0.67-2.64), FT3 5.21±0.96pmol/l (range: 3.47-9.75); FT4 1.35±0.4 ng/ml (range: 0.52-2.57). Malnutrition-inflammation complex syndrome prevalence was 53.9%; 11.7% presented low FT3 levels. Serum T3 and FT3 concentrations inversely correlated with Malnutritition-Inflammation Score (MIS), while FT4 correlated positively with Malnutrition-Inflammation Score. In the linear regression analysis, low FT3 was associated with IL-6 (ß= 0.265, p=.031), C-reactive protein (CRP) (ß= -0.313, p=.018) and albumin (ß= 0.276, p=.002). CONCLUSION: Low T3 and FT3 levels are correlated with malnutrition and inflammation parameters. Malnutrition-inflammation complex syndrome can affect serum concentrations of thyroid hormones.

A genome-wide assessment of adrenocorticotropin action in the Y1 mouse adrenal tumor cell line.

This report summarizes the genome-wide effects of ACTH on transcript accumulation in mouse adrenal Y1 cells and the relative contributions of the cAMP-, protein kinase C- and Ca(2+)-dependent signaling pathways to these actions of the hormone. ACTH affected the accumulation of 1386 transcripts, a much larger number than previously appreciated. The cAMP signaling pathway accounted for approximately 56% of the ACTH effects whereas the protein kinase C- and Ca(2+)-dependent pathways made smaller contributions to ACTH action. Approximately 38% of the ACTH-affected transcripts could not be assigned to these signaling pathways and thus represent candidates for regulation via other mechanisms. The set of ACTH-regulated transcripts included clusters with functions in steroid metabolism, cell proliferation and alternative splicing. Collectively, our results suggest that Y1 adrenal cells undergo extensive remodeling upon prolonged stimulation with ACTH. The functional implications of ACTH on alternative splicing are explored.

Thyroid disorders among dialysis patients / Alteraciones tiroideas en pacientes en diálisis

The thyroid gland and the kidney are closely related. Thyroid hormones (TH) contribute to the homeostasis of the human being through complex interactions of fluids and electrolytes, protein synthesis, etc. The effects on the kidney of TH may be pre renal or direct actions. Decreasing glomerular filtration (GF) this balance especially in advanced stages of chronic kidney disease (CKD) is altered. CKD is linked to alterations in TH levels and/or metabolism, resulting in a high prevalence of subclinical hypothyroidism and low free triiodothyronine (FT3) syndrome. These alterations are linked in micro inflammation, endothelial damage, cardiac abnormalities, and high mortality. In this study, we describe the most common thyroid abnormalities reported in CKD with dialytic stage approach.

La glándula tiroides y el riñón están estrechamente relacionados. Las hormonas tiroideas (HT) contribuyen en la homeostasis del ser humano a través de complejas interacciones de líquidos y electrolitos, síntesis de proteínas, etc. Los efectos de las HT sobre el riñón pueden ser pre-renales o directos. Está demostrado que al disminuir la filtración glomerular (FG) se altera este equilibrio, sobre todo en estadios avanzados de la enfermedad renal crónica (ERC). La ERC está vinculada con alteraciones en los niveles de HT y/o su metabolismo, lo que resulta en una alta prevalencia de hipotiroidismo subclínico y el síndrome de T3 libre baja. Estas alteraciones están relacionadas con micro inflamation, daño endotelial, alteraciones cardiacas y alta mortalidad. El presente estudio, describe las alteraciones tiroideas más frecuentes reportadas en ERC con enfoque en la etapa dialítica.

Global profiles of gene expression induced by adrenocorticotropin in Y1 mouse adrenal cells.

ACTH regulates the steroidogenic capacity, size, and structural integrity of the adrenal cortex through a series of actions involving changes in gene expression; however, only a limited number of ACTH-regulated genes have been identified, and these only partly account for the global effects of ACTH on the adrenal cortex. In this study, a National Institute on Aging 15K mouse cDNA microarray was used to identify genome-wide changes in gene expression after treatment of Y1 mouse adrenocortical cells with ACTH. ACTH affected the levels of 1275 annotated transcripts, of which 46% were up-regulated. The up-regulated transcripts were enriched for functions associated with steroid biosynthesis and metabolism; the down- regulated transcripts were enriched for functions associated with cell proliferation, nuclear transport and RNA processing, including alternative splicing. A total of 133 different transcripts, i.e. only 10% of the ACTH-affected transcripts, were represented in the categories above; most of these had not been described as ACTH-regulated previously. The contributions of protein kinase A and protein kinase C to these genome-wide effects of ACTH were evaluated in microarray experiments after treatment of Y1 cells and derivative protein kinase A-defective mutants with pharmacological probes of each pathway. Protein kinase A-dependent signaling accounted for 56% of the ACTH effect; protein kinase C-dependent signaling accounted for an additional 6%. These results indicate that ACTH affects the expression profile of Y1 adrenal cells principally through cAMP- and protein kinase A- dependent signaling. The large number of transcripts affected by ACTH anticipates a broader range of actions than previously appreciated.

Corticotropin (ACTH) regulates alternative RNA splicing in Y1 mouse adrenocortical tumor cells.

The stimulatory effect of ACTH on gene expression is well documented and is thought to be a major mechanism by which ACTH maintains the functional and structural integrity of the gland. Previously, we showed that ACTH regulates the accumulation of over 1200 transcripts in Y1 adrenal cells, including a cluster with functions in alternative splicing of RNA. On this basis, we postulated that some of the effects of ACTH on the transcription landscape of Y1 cells are mediated by alternative splicing. In this study, we demonstrate that ACTH regulates the alternative splicing of four transcripts - Gnas, Cd151, Dab2 and Tia1. Inasmuch as alternative splicing potentially affects transcripts from more than two-thirds of the mouse genome, we suggest that these findings are representative of a genome-wide effect of ACTH that impacts on the mRNA and protein composition of the adrenal cortex.

Photoactivated polycationic bioactive chitosan nanoparticles inactivate bacterial endotoxins.

INTRODUCTION: The current root canal disinfection protocols fail to markedly inactivate bacterial endotoxins from infected root dentin. This study aimed to evaluate the ability of antibacterial photodynamic therapy with chitosan-conjugated rose bengal nanoparticles (CSRBnps) to selectively inactivate endotoxins/lipopolysaccharides (LPSs). METHODS: Antimicrobial agents such as calcium hydroxide (Ca[OH]2), chitosan nanoparticles (CSnps), CSRBnps, and methylene blue (MB) were assessed for their ability to neutralize LPSs obtained from Pseudomonas aeruginosa in a time-dependent interaction with/without photoactivation (20 and 40 J/cm(2)). The inflammatory potential of the treated/untreated LPSs was assessed on macrophage cells (RAW 267.4) using nitric oxide- and enzyme-linked immunosorbent assay (tumor necrosis factor α and interleukin-6 expression)-based analysis. These antimicrobials were tested directly on macrophage cells for cytotoxicity using the mitochondrial activity assay and light microscopy. The data were analyzed using 1-way analysis of variance and the Tukey test. RESULTS: CSnps were least effective in LPS inactivation. Interluekin-6 expression was reduced only with CSRBnp treatment. CSnps and CSRBnps were completely nontoxic, and MB showed slight toxicity to macrophage cells. Ca(OH)2 was highly cytotoxic (P < .005) even at 30 minutes of exposure. CSRBnps and MB with/without photoactivation significantly inactivated LPSs with reduced nitric oxide and tumor necrosis factor α expression (P < .05). Cell death and detachment after Ca(OH)2 treatment resulted in complete absence of all 3 inflammatory markers. CONCLUSIONS: Photodynamically activated CSRBnps caused significant inactivation of endotoxins and the subsequent reduction of all tested inflammatory markers from activated macrophages. Antimicrobial CSRBnps in combination with photodynamic therapy showed the potential to effectively inactivate bacterial endotoxins.

Prevalencia del Síndrome de Combinación y sus factores asociados en la Clínica de la Facultad de Odontología de la Universidad de Cuenca, periodo 2015-2016 / Prevalence of Combination Syndrome and associated factors at Clínica de la Facultad de Odontología, Universidad de Cuenca, 2015-2016

INTRODUCCIÓN: Un conjunto de cambios patológicos en el sistema estomatognático frente a la pre-sencia de una prótesis total maxilar y una prótesis parcial removible mandibular han sido descritos como el Síndrome de Combinación. El objetivo de este estudio fue determinar la prevalencia del sín-drome y si factores como la edad, el sexo o el tipo de edentulismo mandibular están asociados al mis-mo en los pacientes de la Clínica de la Facultad de Odontología de la Universidad de Cuenca período 2015-2016. MÉTODOS: Estudio observacional,transversal, descriptivo y de correlación. La muestra estuvo cons-tituida por 312 pacientes edéntulos. Se estableció la presencia del síndrome de combinación cuando un paciente presentaba un mínimo de tres signos asociados. Se determinó la prevalencia del síndro-me de combinación en la muestra y la asociación entre las variables sexo, edad, tipo de edentulismo con la presencia de síndrome de combinación. RESULTADOS: La prevalencia del SC resultó en un 21.8%. Padecer de edentulismo parcial clase I de Kennedy (3.6 veces mayor probabilidad que los otros tipos de edentulismo) y ser adulto mayor de 60 años (1.8 veces mayor probabilidad), tuvieron asociación con el desarrollo del síndrome de Combinación. CONCLUSIÓN: La prevalencia de SC es del 21.8%, la progresión de la edad y el edentulismo parcial clase I de Kennedy conducen a una mayor probabilidad para desarrollar el síndrome de combinación y deben evaluarse como factores de riesgo.(au)

BACKGROUND: The set of pathological changes in the stomatognathic system in the presence of a total maxillary prosthesis and a removable partial mandibular prosthesis has been described as the Combi-nation Syndrome. The main purpose of this study was to determine its prevalence and if features such as age, sex or type of mandibular edentulism are associated with the syndrome in the patients of the Odontologic Clinic of the of Faculty of Dentistry, Universidad de Cuenca during the academic period 2015-2016. METHODS: Cuantitative, cross sectional descriptive and correlational study. The sample consisted of 312 edentulous patients. The diagnosis of the syndrome in this study was determined by the presence of a minimum of three associated signs. We determine the prevalence of combiantion syndrome and the as-sociation between sex, age and edentulism type and combination syndrome. RESUlTS: The prevalence of combination syndrome is 21.8%. Adults 60 years or older (1.8 times higher probability) and patients who suffer from Kennedy class I partial edentulism (3.6 times higher probability than other types of edentulism), have a higher probability for developing the syndrome. CONClUSION: The prevalence of CS was 21.8%, age and suffering from Kennedy class I partial edentulism should be considered as risk factors for developing the syndrome.(au)

A polymorphic form of steroidogenic factor-1 is associated with adrenocorticotropin resistance in y1 mouse adrenocortical tumor cell mutants.

ACTH resistance in mutant derivatives of the Y1 mouse adrenocortical tumor cell line results from a defect that affects the activity of steroidogenic factor-1 (SF1), thereby preventing the expression of the melanocortin-2 receptor. In this report, we show that the SF1 genes in ACTH-resistant mutants differ from the gene in ACTH-responsive Y1 cells by two base changes-one that changes an Ala to Ser at codon 172, and one in the third position of codon 3 that does not affect the protein sequence. Furthermore, several of the mutants contain multiple copies of this alternate SF1 gene (SF1(S172)) on acentric chromosome fragments. The SF1(S172) allele represents a polymorphism rather than a spontaneous mutation because the two SF1 alleles can be traced to the hybrid mouse strain (C57L/J x A/HeJ) from which the original adrenal tumor was derived. The SF1(A172) allele also is found in C57Bl/6J and C57Bl/10J mice, whereas the SF1(S172) allele also is found in C3H/HeJ and DBA/2J mice. The two forms of SF1 had only modest differences in activity suggesting that the SF1 polymorphism per se is not directly responsible for ACTH resistance. Our results indicate that the SF1(S172) allele is a marker of ACTH resistance in this family of adrenocortical tumor cells.

A polymorphic form of steroidogenic factor 1 associated with ACTH receptor deficiency in mouse adrenal cell mutants.

We have described a family of adrenocortical tumor cell mutants (including clones OS3, Y6, and 10r9) that are resistant to ACTH because they fail to express the gene encoding the ACTH receptor (MC2R). The MC2R deficiency results from a mutation that impairs the activity of the nuclear receptor steroidogenic factor 1 (SF1) at the MC2R promoter. In this report, we show that ACTH resistance in the mutant clones is associated with a Sf1 gene that has Ser at codon 172 instead of Ala. In two of the three mutant clones, this Sf1 allele is amplified together with flanking DNA from chromosome 2 that includes the genes encoding germ cell nuclear factor and the beta-type proteosome subunit Psmb7. SF1(A172) and SF1(S172) exhibit little or no difference in transcriptional activity in SF1-dependent reporter gene assays, suggesting that SF1(S172) per se is not directly responsible for the loss of MC2R expression. Instead, the Sf1(S172) allele appears to be a marker of ACTH resistance in this family of adrenocortical tumor cell mutants, possibly reflecting the activity of a neighboring gene.

A novel function for the competence inducing peptide, XIP, as a cell death effector of Streptococcus mutans.

In Streptococcus mutans, ComX, an alternative sigma factor, drives the transcription of the 'late-competence genes' required for genetic transformation. ComX activity is modulated by inputs from two signaling pathways, ComDE and ComRS, that respond to the competence-stimulating peptide (CSP) and the SigX-inducing peptide (XIP), respectively. In particular, the comRS, encoding the ComR regulatory protein and the ComS precursor to XIP, functions as the proximal regulatory system for ComX activation. Here, we investigated the individual and combinatorial effects of CSP and XIP on genetic transformation and cell killing of S. mutans. Our transformation results confirm the recent reports by Mashburn-Warren et al. and Desai et al. that XIP functions optimally in a chemically defined medium, whereas its activity is inhibited when cells are grown in complex medium. Using tandem mass spectrometry (MS/MS) fragmentation, a drastic reduction in XIP levels in ComX-deficient cultures were observed, suggesting a ComX-mediated positive feedback mechanism for XIP synthesis. Our evaluation of cell viability in the presence of 10 µM XIP resulted in killing nearly 82% of the population. The killing activity was shown to be dependent on the presence of comR/S and comX. These results suggest a novel role for XIP as a compelling effector of cell death. This is the first report that demonstrates a role for XIP in cell killing.

Contributions of steroidogenic factor 1 to the transcription landscape of Y1 mouse adrenocortical tumor cells.

The contribution of steroidogenic factor 1 (SF-1) to the gene expression profile of Y1 mouse adrenocortical cells was evaluated using short hairpin RNAs to knockdown SF-1. The reduced level of SF-1 RNA was associated with global changes that affected the accumulation of more than 2000 transcripts. Among the down-regulated transcripts were several with functions in steroidogenesis that were affected to different degrees--i.e., Mc2r>Scarb1>Star≥Hsd3b1>Cyp11b1. For Star and Cyp11b1, the different levels of expression correlated with the amount of residual SF-1 bound to the proximal promoter regions. The knockdown of SF-1 did not affect the accumulation of Cyp11a1 transcripts even though the amount of SF-1 bound to the proximal promoter of the gene was reduced to background levels. Our results indicate that transcripts with functions in steroidogenesis vary in their dependence on SF-1 for constitutive expression. On a more global scale, SF-1 knockdown affects the accumulation of a large number of transcripts, most of which are not recognizably involved in steroid hormone biosynthesis.

SF1 polymorphisms in the mouse and steroidogenic potential.

ACTH-resistance in four mutant derivatives of a mouse adrenocortical tumor cell line results from a defect that reduces the activity of steroidogenic factor-1 (SF1) thereby preventing expression of the ACTH receptor and other SF1-dependent genes. The SF1 genes from these mutants contain a sequence difference that changes an Ala to Ser at codon 172. Steroidogenic factor-1(S172) represents a polymorphism rather than a spontaneous mutation since the two forms of SF1, SF1(A172), and SF1(S172), can be traced to the hybrid mouse strain (C57L/J x A/HeJ) from which the original adrenal tumor was derived. The SF1(S172) allele is amplified in three of the four mutant clones together with the neighboring genes germ cell nuclear factor and LIM homeobox2. The two forms of SF1 had only modest differences in transcriptional activity in reporter gene assays, suggesting that the SF1 polymorphism per se is not directly responsible for the loss of mc2r expression. Rather, ACTH resistance in this family of adrenocortical tumor cell mutants may be due to a closely linked gene on the SF1(S172) allele. Mouse strains with reportedly high steroidogenic capacity (C57Bl/6J, C57Bl/10J) also have the SF1(A172) allele while mouse strains with low steroidogenic capacity (C3H/HeJ, DBA/2J) have the SF1(S172) allele. These latter observations suggest that the two SF1 alleles also may be markers of steroidogenic potential among mouse strains.