Multivariate curve resolution-based data fusion approaches applied in 1H NMR metabolomic analysis of healthy cohorts.

BACKGROUND: Metabolomics plays a critical role in deciphering metabolic alterations within individuals, demanding the use of sophisticated analytical methodologies to navigate its intricate complexity. While many studies focus on single biofluid types, simultaneous analysis of multiple matrices enhances understanding of complex biological mechanisms. Consequently, the development of data fusion methods enabling multiblock analysis becomes essential for comprehensive insights into metabolic dynamics. RESULTS: This study introduces a novel guideline for jointly analyzing diverse metabolomic datasets (serum, urine, metadata) with a focus on metabolic differences between groups within a healthy cohort. The guideline presents two fusion strategies, 'Low-Level data fusion' (LLDF) and 'Mid-Level data fusion' (MLDF), employing a sequential application of Multivariate Curve Resolution with Alternating Least Squares (MCR-ALS), linking the outcomes of successive analyses. MCR-ALS is a versatile method for analyzing mixed data, adaptable at various stages of data processing-encompassing resonance integration, data compression, and exploratory analysis. The LLDF and MLDF strategies were applied to 1H NMR spectral data extracted from urine and serum samples, coupled with biochemical metadata sourced from 145 healthy volunteers. SIGNIFICANCE: Both methodologies effectively integrated and analysed multiblock datasets, unveiling the inherent data structure and variables associated with discernible factors among healthy cohorts. While both approaches successfully detected sex-related differences, the MLDF strategy uniquely revealed components linked to age. By applying this analysis, we aim to enhance the interpretation of intricate biological mechanisms and uncover variations that may not be easily discernible through individual data analysis.

A different gut microbiome linked to inflammation found in cirrhotic patients with and without hepatocellular carcinoma.

INTRODUCTION AND AIM: A pro-oncogenic intestinal microbiome was observed in murine models; however, no specific microbiome in patients with hepatocellular carcinoma (HCC) has been reported. We aimed to compare the gut microbiome found in cirrhotic patients with or without HCC. MATERIALS AND METHODS: From 407 patients with Child Pugh A/B cirrhosis prospectively followed, 25 with HCC (cases) were matched with 25 without HCC (wo-HCC) in a 1:1 ratio according to age, gender, etiology, Child Pugh and severity of portal hypertension. In addition, results were also compared with 25 healthy subjects. Fecal stool samples were sequenced for the V3-V4 region of the microbial 16S rRNA (Illumina MiSeq Platform). Plasma cytokines were quantified including interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). RESULTS: We found a differential abundance in family members of Firmicutes with a 3-fold increase of Erysipelotrichaceae and a 5-fold decrease in family Leuconostocaceae in HCC when compared to wo-HCC controls. Genus Fusobacterium was found to be 5-fold decreased in HCC vs wo-HCC. The ratio bacteriodes/prevotella was increased in HCC. Three operational taxonomic units (OTUs), genus Odoribacter and Butyricimonas were more abundant in HCC, whereas a decreased abundance in Lachnospiraceae family genus Dorea was observed in HCC patients. A Random Forest model trained with differential abundant taxa correctly classified HCC individuals. This pattern was associated with an inflammatory milieu with a putative increased activation of NOD-like receptor pathways. CONCLUSION: We found a pattern of microbiome linked to inflammation that could be potentially useful as HCC biomarker after follow-up validation studies.

Human Microbiota of the Argentine Population- A Pilot Study.

The human microbiota is the collection of microorganisms living in or on the human body. An imbalance or dysbiosis in these microbial communities can be associated with a wide variety of human diseases (Petersen and Round, 2014; Pham and Lawley, 2014; Zaura et al., 2014). Moreover, when the microbiota of the same body sites is compared between different healthy individuals, specific microbial community features are apparent (Li et al., 2012; Yatsunenko et al., 2012; Oh et al., 2014; Relman, 2015). In addition, specific selective pressures are found at distinct body sites leading to different patterns in microbial community structure and composition (Costello et al., 2009; Consortium, 2012b; Zhou et al., 2013). Because of these natural variations, a comprehensive characterization of the healthy microbiota is critical for predicting alterations related to diseases. This characterization should be based on a broad healthy population over time, geography, and culture (Yatsunenko et al., 2012; Shetty et al., 2013; Leung et al., 2015; Ross et al., 2015). The study of healthy individuals representing different ages, cultural traditions, and ethnic origins will enable to understand how the associated microbiota varies between populations and respond to different lifestyles. It is important to address these natural variations in order to later detect variations related to disease.

A thin-layer chromatography autographic method for the detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system.

INTRODUCTION: The PhoP-PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. OBJECTIVE: To develop a TLC bioautography to detect inhibitors of the PhoP-PhoQ regulatory system in complex matrixes. METHODS: The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. RESULTS: A bioautographic assay suitable for the localisation of inhibitors of the PhoP-PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP-PhoQ system. CONCLUSION: A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP-PhoQ regulatory system from a hydrolysed extract.

Unsaturated long chain free fatty acids are input signals of the Salmonella enterica PhoP/PhoQ regulatory system.

The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg(2+) limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the ß-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.

Two component systems in the spatial program of bacteria.

Despite being considered a relatively simple form of life, bacteria have revealed a high degree of structural organization, with the spatial destination of their components precisely regulated within the cell. Nevertheless, the primary signals that dictate differential distribution of cellular building blocks and physiological processes remain in most cases largely undisclosed. Signal transduction systems are no exception within this three-dimensional organization and two-component systems (TCS) involved in controlling cell division, differentiation, chemotaxis and virulence show specific and/or dynamic localization, engaging in the spatial program of the bacterial cell.

Cytolocalization of the PhoP response regulator in Salmonella enterica: modulation by extracellular Mg2+ and by the SCV environment.

The PhoP/PhoQ two-component system plays an essential role regulating numerous virulence phenotypes in Salmonella enterica. Previous work showed that PhoQ, the sensor protein, switches between the kinase- and the phosphatase-dominant state in response to environmental Mg2+ availability. This switch defines the PhoP phosphorylation status and, as a result, the transcriptional activity of this regulator. In this work, using the FlAsH labelling technique, we examine PhoP cytolocalization in response to extracellular Mg2+ limitation in vitro and to the Salmonella-containing vacuole (SCV) environment in macrophage cells. We demonstrate that in these PhoP/PhoQ-inducing environments PhoP displays preferential localization to one cell pole, while being homogeneously distributed in the bacterial cytoplasm in repressing conditions. Polar localization is lost in the absence of PhoQ or when a non-phosphorylatable PhoP(D52A) mutant is expressed. However, when PhoP transcriptional activation is achieved in a Mg2+- and PhoQ-independent manner, PhoP regains asymmetric polar localization. In addition, we show that, in the analysed conditions, PhoQ cellular distribution does not parallel PhoP location pattern. These findings reveal that PhoP cellular location is dynamic and conditioned by its environmentally defined transcriptional status, showing a new insight in the PhoP/PhoQ system mechanism.

High TGFbeta1, estrogen receptor, and aromatase gene expression in a large cell calcifying sertoli cell tumor (LCCSCT): implications for the mechanism of oncogenesis.

Large cell calcifying Sertoli cell tumors (LCCSCT) are associated with Carney complex and Peutz-Jeghers syndrome. The mechanisms linking these 2 genetic defects to the genesis of this tumor are obscure. Studies of CYP19 (aromatase) and transforming growth factor (TGF)-beta1 messenger RNA (mRNA) abundance, estrogen receptor (ER), TGFbeta1, and TGFbeta type II receptor (R) immunochemistry were carried out in the testis of a patient with this tumor to gain information on possible mechanisms of cell tumor development. Testicular tissue of a prepubertal patient, collected at gonadectomy, was separated into 2 macroscopically distinct fractions: tumoral nodules (Tu) and extratumoral, normal-looking testicular tissue (ExTu). The patient was a 9.5-year-old boy with a 5-year history of bilateral gynecomastia (Tanner stage 4), no pubic hair, incipient genital development, and bilateral testicular nodules. Multiple pigmented lesions of the skin were present. Bilateral mammectomy and gonadectomy was performed. RNA was extracted from Tu and ExTu for semiquantitative reverse transcriptase-polymerase chain reaction of CYP19 and TGFbeta1. Protein expression of ER, TGFbeta1, and TGFbeta type II R in Tu and ExTu was detected by immunohistochemistry. Cell proliferation was estimated by Ki-67 antigen immunochemistry and apoptosis using a modified TUNEL assay. Mean expression of aromatase and TGFbeta1 mRNAs in Tu was 6- and 2.3-fold higher than in ExTu, respectively (P<0.05). Tumoral cells exhibited ER staining with a predominant extranuclear localization. Positive staining of Sertoli cells in Tu was higher than in ExTu. TGFbeta1 immunostaining of the interstitial cells in Tu was higher than in ExTu. TGFbeta type II R immunostaining was detected in most Sertoli and interstitial cells, but intensity in ExTu was lower than in Tu. No significant difference was detected in the proliferation index, but in Tu, the percentage of Sertoli cells in apoptosis (1.4%) was significantly lower (P<0.01) than in ExTu (14.0%). The following hypothesis is proposed. The congenital gene defects of Carney complex or of Peutz-Jeghers syndrome might trigger a cascade of intracellular events that leads to overexpression of aromatase in Sertoli cells, favoring the development of a LCCSCT. At some point in the evolution of the disease, a mutational event might induce a higher expression of the ER. Also, TGFbeta1 protein expression is increased in neighboring cells. In this environment, TGFbeta1 might switch from tumor suppressor to oncogenic factor and, along with estrogen-ER complexes, might favor tumor progression by inhibiting apoptosis.

Apoptosis and proliferation of human testicular somatic and germ cells during prepuberty: high rate of testicular growth in newborns mediated by decreased apoptosis.

Programmed cell death and proliferation are evolutionary conserved processes that play a major role during normal development and homeostasis. In the testis, during the fetal and newborn periods, they might determine final adult size and fertility potential. In the present study, we have measured the relative number of testicular cells in apoptosis and in active proliferation in the seminiferous cords and in the interstitium, at different age periods of prepubertal testicular development in humans. Testes from 44 prepubertal subjects without endocrine and metabolic abnormalities were collected at necropsy. They were divided in three age groups (Gr): Gr 1, newborn (1- to 21-d-old neonates), n = 18, mean (+/-SD) age 0.3 +/- 0.23 months; Gr 2, post natal activation (1- to 6-month-old infants), n = 13, mean age 3.93 +/- 1.90 months; and Gr 3, early childhood period (1- to <6-yr-old boys), n = 13, mean age 31.5 +/- 18.9 months. Apoptosis was detected in 5- microm tissue sections using a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and cell proliferation was assessed by Ki-67 immunohistochemistry. Evaluation of apoptosis was confirmed by estimation of active caspase-3. Mean (+/-SD) testicular weight was 0.38 +/- 0.20, 0.54 +/- 0.35, and 0.51 +/- 0.11 g in Gr 1, Gr 2, and Gr 3, respectively. In Gr 1, there was a significant positive correlation between age and testis weight (P = 0.02). Mean (+/-SD) germ cell apoptotic index, AI, (% of apoptotic cells out of total cell number) was 15.0 +/- 6.60, 27.0 +/- 8.80 and 33.4 +/- 11.4 in Gr 1, Gr 2, and Gr 3, respectively. In Sertoli cells, it was 6.60 +/- 4.07, 22.0 +/- 14.0 and 27.5 +/- 19.8, respectively. In interstitial cells, it was 10.2 +/- 6.38, 18.0 +/- 6.70 and 25.7 +/- 15.5, respectively. In the three types of cells, AI in Gr 1 was significantly lower than in Gr 2 or Gr 3 (P < 0.05). Mean (+/-SD) germ cell proliferation index, PI, was 18.6 +/- 13.0, 10.0 +/- 6.50 and 10.9 +/- 6.24% in Gr 1, Gr 2, and Gr 3, respectively. In Sertoli cells and in interstitial cells PI was similar in the three age groups. The PI/AI ratio was used to compare relative differences among age groups. The PI/AI ratio of germ cells, Sertoli cells and interstitial cells in Gr 1 was significantly higher than in Gr 2 or Gr 3 (P < 0.05). It is concluded that, in normal subjects, there is a vigorous growth of the testis during the newborn period with subsequent stabilization during the first years of prepuberty. This cell growth seems to be mainly mediated by decreased apoptosis. The factors that modulate apoptosis of testicular cells are not known, but it is remarkable that this change takes place before the testosterone peak of the post natal gonadal activation of the first trimester of life. These changes taking place during the newborn period might be important to define testicular function in adults.