Osteocalcin associates with bone mineral density and VDR gene polymorphisms in type 1 and type 2 diabetes.

Objectives: Bone metabolism is impaired in diabetes mellitus (DM). Our objective is to evaluate the association of bone turnover markers (BTM) and vitamin D receptor (VDR) gene polymorphisms with bone mineral density (BMD) in DM type 1 (T1D) and DM type 2 (T2D). Methods: A total of 165 patients (53 T1D and 112 T2D) were enrolled. BMD was measured by dual-energy X-ray absorptiometry (DEXA). Plasma osteocalcin (OC), beta-CrossLaps (ß-CTX) and N-amino terminal propeptide of type I collagen (P1NP) and VDR gene polymorphisms were evaluated. Results: Participants were 53 T1D (41 years [31-48]) and 112 T2D (60 years [51-66]). BMD were not statistically different between the groups. OC (p<0.001) and P1NP levels (p<0.001) were higher in patients with T1D. The areas under the curve for the prediction of bone pathology were 0.732 (p=0.038) for OC in T1D and 0.697 (p=0.007) in T2D. A significant association was found between lower lumbar BMD and the A allele of BsmI (p=0.03), the A allele of ApaI (p=0.04) and the allele C of the Taql (p=0.046). Also, a significant correlation was found with higher OC levels and the G allele of BsmI (p=0.044), C allele of ApaI (p=0.011), T allele of Taql (p=0.006) and with C allele of FokI (p=0.004). Conclusions: The high negative predictive value of the cut-off point for OC suggests that could be useful in excluding the risk suffering bone loss, allowing offering a personalized clinical approach to prevent this pathology.

The response to fasting and refeeding reveals functional regulation of lipoprotein lipase proteoforms.

Lipoprotein lipase (LPL) is responsible for the intravascular catabolism of triglyceride-rich lipoproteins and plays a central role in whole-body energy balance and lipid homeostasis. As such, LPL is subject to tissue-specific regulation in different physiological conditions, but the mechanisms of this regulation remain incompletely characterized. Previous work revealed that LPL comprises a set of proteoforms with different isoelectric points, but their regulation and functional significance have not been studied thus far. Here we studied the distribution of LPL proteoforms in different rat tissues and their regulation under physiological conditions. First, analysis by two-dimensional electrophoresis and Western blot showed different patterns of LPL proteoforms (i.e., different pI or relative abundance of LPL proteoforms) in different rat tissues under basal conditions, which could be related to the tissue-specific regulation of the enzyme. Next, the comparison of LPL proteoforms from heart and brown adipose tissue between adults and 15-day-old rat pups, two conditions with minimal regulation of LPL in these tissues, yielded virtually the same tissue-specific patterns of LPL proteoforms. In contrast, the pronounced downregulation of LPL activity observed in white adipose tissue during fasting is accompanied by a prominent reconfiguration of the LPL proteoform pattern. Furthermore, refeeding reverts this downregulation of LPL activity and restores the pattern of LPL proteoforms in this tissue. Importantly, this reversible proteoform-specific regulation during fasting and refeeding indicates that LPL proteoforms are functionally diverse. Further investigation of potential differences in the functional properties of LPL proteoforms showed that all proteoforms exhibit lipolytic activity and have similar heparin-binding affinity, although other functional aspects remain to be investigated. Overall, this study demonstrates the ubiquity, differential distribution and specific regulation of LPL proteoforms in rat tissues and underscores the need to consider the existence of LPL proteoforms for a complete understanding of LPL regulation under physiological conditions.

Aspergillosis by cryptic Aspergillus species: A case series and review of the literature.

BACKGROUND: The cryptic Aspegillus species are rare, these microorganisms are usually more resistant to common antifungal therapies. Therefore, a correct identification is important when evaluating the impact of such species in aspergillosis. AIMS: We aimed to describe the frequency, clinical and microbiological characteristics, and the outcomes of those cases of aspergillosis caused by cryptic species in a tertiary hospital. METHODS: We retrospectively identified all microbiologically documented cases of aspergillosis between January 2013 and December 2018. Definitive species identification of clinically significant isolates was achieved via sequencing methods. The polymerase chain reaction (PCR) products were sequenced, and the results obtained were compared to sequences deposited in GenBank. Antifungal susceptibility testing was performed using the Sensititre® YeastOne® panel. RESULTS: A total of 679 Aspergillus isolates were recovered from 489 patients, of which 109 were clinically relevant. Ten (9.2%) isolates were identified as cryptic species: Aspergillus arcoverdensis (2), Aspergillus lentulus (2), Aspergillus ellipticus (2), Aspergillus alliaceus (1), Aspergillus nomius (1), Aspergillus tubingensis (1) and Aspergillus montevidensis (1). Most patients already suffered some type of immunosuppression. Half of these patients had required intensive care before the infection showed up, and most of them had a pulmonary infection. Mortality at the 100-day follow-up was 40%. Antifungal susceptibility testing was performed on three of the isolates (A. arcoverdensis, A. tubingensis and A. nomius), which showed high minimum inhibitory concentrations (MIC) for azoles and amphotericin B. CONCLUSIONS: The frequency of cryptic species in our centre was 9.2%. Most patients had some degree of immunosuppression, and the mortality rate was 40%.

Dietary Patterns in Pregnancy and Biomarkers of Oxidative Stress in Mothers and Offspring: The NELA Birth Cohort.

Background: Although adherence to the Mediterranean and antioxidant-rich diets during pregnancy is suggested to improve maternal-fetal health by reducing oxidative stress, yet there is no study available. Objective: We examined whether maternal dietary patterns in pregnancy impact the biomarkers of oxidative stress in mothers and their offspring. Methods: Study population included 642 mothers and 335 newborns of the "Nutrition in Early Life and Asthma" (NELA) birth cohort. Maternal diet during pregnancy was assessed by a validated food frequency questionnaire and a priori-defined dietary indices (relative Mediterranean Diet [rMED], alternative Mediterranean Diet [aMED], Dietary Approach to Stop Hypertension [DASH], Alternate Healthy Index [AHEI], and AHEI-2010) were calculated. Biomarkers measured were: hydroperoxides, carbonyl groups, and 8-hydroxydeoxyguanosine (8OHdG) determined in maternal blood and newborn cord blood, and urinary maternal and offspring 15-F2t-isoprostane. Multivariate linear regression models were performed. Results: Maternal rMED score was inversely associated with the maternal levels of 8OHdG at mid-pregnancy (beta per 1-point increase = -1.61; 95% CI -2.82, -0.39) and the newborn levels of hydroperoxides (beta per 1-point increase = -4.54; 95% CI -9.32, 0.25). High vs. low maternal rMED score was marginally associated with the decreased levels of 8OHdG in newborns (beta = -9.17; 95% CI -19.9, 1.63; p for trend 0.079). Maternal DASH score tended to be inversely associated with maternal urinary 15-F2t-isoprostane (beta per 1-point increase = -0.69; 95% CI, -1.44, 0.06). High vs. low maternal AHEI score was associated with reduced offspring urinary levels of 15-F2t-isoprostane (beta = -20.2; 95% CI -38.0, -2.46; p for trend 0.026). Conclusion: These results suggest that maternal adherence to healthy dietary patterns during pregnancy may reduce DNA damage and lipid oxidation in mothers and offspring.

Aspergillosis by cryptic Aspergillus species: A case series and review of the literature

Background:The cryptic Aspegillus species are rare, these microorganisms are usually more resistant to common antifungal therapies. Therefore, a correct identification is important when evaluating the impact of such species in aspergillosis.Aims:We aimed to describe the frequency, clinical and microbiological characteristics, and the outcomes of those cases of aspergillosis caused by cryptic species in a tertiary hospital.Methods:We retrospectively identified all microbiologically documented cases of aspergillosis between January 2013 and December 2018. Definitive species identification of clinically significant isolates was achieved via sequencing methods. The polymerase chain reaction (PCR) products were sequenced, and the results obtained were compared to sequences deposited in GenBank. Antifungal susceptibility testing was performed using the Sensititre® YeastOne® panel.Results:A total of 679 Aspergillus isolates were recovered from 489 patients, of which 109 were clinically relevant. Ten (9.2%) isolates were identified as cryptic species: Aspergillus arcoverdensis (2), Aspergillus lentulus (2), Aspergillus ellipticus (2), Aspergillus alliaceus (1), Aspergillus nomius (1), Aspergillus tubingensis (1) and Aspergillus montevidensis (1). Most patients already suffered some type of immunosuppression. Half of these patients had required intensive care before the infection showed up, and most of them had a pulmonary infection. Mortality at the 100-day follow-up was 40%. Antifungal susceptibility testing was performed on three of the isolates (A. arcoverdensis, A. tubingensis and A. nomius), which showed high minimum inhibitory concentrations (MIC) for azoles and amphotericin B.Conclusions:The frequency of cryptic species in our centre was 9.2%. Most patients had some degree of immunosuppression, and the mortality rate was 40%. (AU)

Antecedentes:Las especies crípticas dentro del género Aspergillus son poco habituales, pero suelen mostrar una mayor resistencia al tratamiento antifúngico convencional. Por tanto, una correcta identificación de la especie es necesaria para evaluar el impacto de estas especies crípticas en el desarrollo de la aspergilosis.Objetivos:El objetivo de este estudio fue describir las características clínicas, epidemiológicas y microbiológicas, así como la evolución clínica, de los casos de aspergilosis por especies crípticas en un hospital de tercer nivel.Métodos:Se analizaron de forma retrospectiva todos los casos documentados de aspergilosis con identificación microbiológica entre enero de 2013 y diciembre de 2018. La identificación definitiva de los aislamientos clínicos se realizó mediante métodos de secuenciación. Los productos de amplificación obtenidos por la reacción en cadena de la polimerasa (PCR) fueron secuenciados, y los resultados se analizaron utilizando la base de datos del GenBank. Para el análisis de susceptibilidad a los antifúngicos de los aislamientos identificados se utilizó el panel Sensititre® YeastOne®.Resultados:Se identificaron un total de 679 aislamientos de Aspergillus de 489 pacientes, de los cuales un total de 109 eran clínicamente relevantes. Diez (9,2%) de los aislamientos correspondían a especies crípticas: Aspergillus arcoverdensis (2), Aspergillus lentulus (2), Aspergillus ellipticus (2), Aspergillus alliaceus (1), Aspergillus nomius (1), Aspergillus tubingensis (1) y Aspergillus montevidensis (1). La mayoría de los pacientes tenían algún tipo de inmunosupresión previa. La mitad de estos pacientes habían requerido de cuidados intensivos antes de la infección, y la mayoría sufría una infección pulmonar. La mortalidad a los 100 días de seguimiento fue del 40%. (AU)

Self-Reported DHA Supplementation during Pregnancy and Its Association with Obesity or Gestational Diabetes in Relation to DHA Concentration in Cord and Maternal Plasma: Results from NELA, a Prospective Mother-Offspring Cohort.

Maternal supplementation of docosahexaenoic acid (DHA) during pregnancy has been recommended due to its role in infant development, but its effect on materno-fetal DHA status is not well established. We evaluated the associations between DHA supplementation in pregnant women with obesity or gestational diabetes mellitus (GDM) and maternal and neonatal DHA status. Serum fatty acids (FA) were analyzed in 641 pregnant women (24 weeks of gestation) and in 345 venous and 166 arterial cord blood samples of participants of the NELA cohort. Obese women (n = 47) presented lower DHA in serum than those lean (n = 397) or overweight (n = 116) before pregnancy. Linoleic acid in arterial cord was elevated in obese women, which indicates lower fetal retention. Maternal DHA supplementation (200 mg/d) during pregnancy was associated with enhanced maternal and fetal DHA levels regardless of pre-pregnancy body mass index (BMI), although higher arterial DHA in overweight women indicated an attenuated response. Maternal DHA supplementation was not associated with cord venous DHA in neonates of mothers with GDM. The cord arteriovenous difference was similar for DHA between GDM and controls. In conclusion, maternal DHA supplementation during pregnancy enhanced fetal DHA status regardless of the pre-pregnancy BMI while GDM may reduce the effect of DHA supplementation in newborns.

Evaluation of Computationally Designed Peptides against TWEAK, a Cytokine of the Tumour Necrosis Factor Ligand Family.

The tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumour necrosis factor ligand family and has been shown to be overexpressed in tumoral cells together with the fibroblast growth factor-inducible 14 (Fn14) receptor. TWEAK-Fn14 interaction triggers a set of intracellular pathways responsible for tumour cell invasion and migration, as well as proliferation and angiogenesis. Hence, modulation of the TWEAK-Fn14 interaction is an important therapeutic goal. The targeting of protein-protein interactions by external agents, e.g., drugs, remains a substantial challenge. Given their intrinsic features, as well as recent advances that improve their pharmacological profiles, peptides have arisen as promising agents in this regard. Here, we report, by in silico structural design validated by cell-based and in vitro assays, the discovery of four peptides able to target TWEAK. Our results show that, when added to TWEAK-dependent cellular cultures, peptides cause a down-regulation of genes that are part of TWEAK-Fn14 signalling pathway. The direct, physical interaction between the peptides and TWEAK was further elucidated in an in vitro assay which confirmed that the bioactivity shown in cell-based assays was due to the targeting of TWEAK. The results presented here are framed within early pre-clinical drug development and therefore these peptide hits represent a starting point for the development of novel therapeutic agents. Our approach exemplifies the powerful combination of in silico and experimental efforts to quickly identify peptides with desirable traits.

Proteomics in Deaths by Drowning: Diagnostic Efficacy of Apolipoprotein A1 and α-1Antitrypsin, Pilot Study.

Drowning is one of the leading causes of death worldwide. The pathophysiology of drowning is complex and, sometimes, interpretation of the circumstances of death in the autopsy becomes the main source of information in its diagnosis. New advances in medical research, such as proteomics, especially in forensic pathology, are still in the development. We proposed to investigate the application of Mass Spectrometry-based technologies, to identify differentially expressed proteins that may act as potential biomarkers in the postmortem diagnosis of drowning. We performed a pilot proteomic experiment with the inclusion of two drowned and two control forensic cases. After applying restrictive parameters, we identified apolipoprotein A1 (ApoA1) and α-1 antitrypsin as differentially expressed between the two diagnostic groups. A validation experiment, with the determination of both proteins in 25 forensic cases (16 drowned and 9 controls) was performed, and we corroborated ApoA1 higher values in the drowning group, whereas α-1 antitrypsin showed lower levels. After adjusting by confounder factors, both remained as predictive independent factors for diagnosis of drowning (p = 0.010 and p = 0.022, respectively). We constructed ROC curves for biomarkers' levels attending at the origin of death and established an ApoA1 cut-off point of 100 mg/dL. Correct classification based on the diagnosis criteria was reached for 73.9% of the cases in a discriminant analysis. We propose apolipoprotein A1 (with our cutoff value for correct classification) and α-1 antitrypsin as valuable biomarkers of drowning. Our study, based on forensic cases, reveals our proteomic approach as a new complementary tool in the forensic diagnosis of drowning and, perhaps, in clinical future implications in drowned patients. However, this is a pilot approach, and future studies are necessary to consolidate our promising preliminary data.

GRP94 promotes brain metastasis by engaging pro-survival autophagy.

BACKGROUND: GRP94 is a glucose-regulated protein critical for survival in endoplasmic reticulum stress. Expression of GRP94 is associated with cellular transformation and increased tumorigenicity in breast cancer. Specifically, overexpression of GRP94 predicts brain metastasis (BM) in breast carcinoma patients with either triple negative or ErbB2 positive tumors. The aim of this study was to understand if microenvironmental regulation of GRP94 expression might be a hinge orchestrating BM progression. METHODS: GRP94 ablation was performed in a BM model BR-eGFP-CMV/Luc-V5CA1 (BRV5CA1) of breast cancer. In vitro results were validated in a dataset of 29 metastases in diverse organs from human breast carcinomas and in BM tissue from tumors of different primary origin. BM patient-derived xenografts (PDXs) were used to test sensitivity to the therapeutic approach. RESULTS: BMs that overexpress GRP94 as well as tumor necrosis factor receptor-associated factor 2 are more resistant to glucose deprivation by induction of anti-apoptotic proteins (B-cell lymphoma 2 and inhibitors of apoptosis proteins) and engagement of pro-survival autophagy. GRP94 ablation downregulated autophagy in tumor cells, resulting in increased BM survival in vivo. These results were validated in a metastasis dataset from human patients, suggesting that targeting autophagy might be strategic for BM prevention. Indeed, hydroxychloroquine treatment of preclinical models of BM from PDX exerts preventive inhibition of tumor growth (P < 0.001). CONCLUSIONS: We show that GRP94 is directly implicated in BM establishment by activating pro-survival autophagy. Disruption of this compensatory fueling route might prevent metastatic growth.

Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol.

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate-glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.

An Integrative Screening Tool of Alcohol Exposure During Early Pregnancy: Combining of the CDT Biomarker with Green Page Questionnaire.

AIMS: In current clinical practice, prenatal alcohol exposure is usually assessed by interviewing the pregnant woman by applying questionnaires. An alternative method for detecting alcohol use is to measure the biomarker carbohydrate-deficient transferrin (CDT). However, few studies measure CDT during pregnancy. This study examines the utility of CDT biomarker in the screening of alcohol exposure during early pregnancy. METHODS: A cohort of 91, first-trimester pregnant women assigned to a public reference maternity hospital, was screened using the Green Page (GP) questionnaire, an environmental exposure tool. CDT levels and other biomarkers of alcohol use were measured and compared with questionnaire data. RESULTS: About 70% of the mothers in the study consumed alcohol during early pregnancy and 22% met high-risk criteria for prenatal exposure to alcohol. CDT measurement showed a statistically significant area under the receiver operating characteristic curve with a value of 0.70. For a value of 0.95% of CDT, a specificity of 93% was observed. The most significant predictors of CDT were the number of binge drinking episodes, women's body mass index and European white race. CONCLUSION: Pregnant women with a CDT value >0.95% would be good candidates for the performance of the GP questionnaire during early pregnancy in order to detect potential high-risk pregnancy due to alcohol exposure.

An Engineered Constitutive Promoter Set with Broad Activity Range for Cupriavidus necator H16.

Well-characterized promoters with variable strength form the foundation of heterologous pathway optimization. It is also a key element that bolsters the success of microbial engineering and facilitates the development of biological tools like biosensors. In comparison to microbial hosts such as Escherichia coli and Saccharomyces cerevisiae, the promoter repertoire of Cupriavidus necator H16 is highly limited. This limited number of characterized promoters poses a significant challenge during the engineering of C. necator H16 for biomanufacturing and biotechnological applications. In this article, we first examined the architecture and genetic elements of the four most widely used constitutive promoters of C. necator H16 (i.e., P phaC1, P rrsC, P j5, and P g25) and established a narrow 6-fold difference in their promoter activities. Next, using these four promoters as starting points and applying a range of genetic modifications (including point mutation, length alteration, incorporation of regulatory genetic element, promoter hybridization, and configuration alteration), we created a library of 42 constitutive promoters, all of which are functional in C. necator H16. Although these promoters are also functional in E. coli, they show different promoter strength and hierarchical rank of promoter activity. Subsequently, the activity of each promoter was individually characterized, using l-arabinose-inducible P BAD promoter as a benchmark. This study has extended the range of constitutive promoter activities to 137-fold, with some promoter variants exceeding the l-arabinose-inducible range of P BAD promoter. Not only has the work enhanced our flexibility in engineering C. necator H16, it presented novel strategies in adjusting promoter activity in C. necator H16 and highlighted similarities and differences in transcriptional activity between this organism and E. coli.

Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories.

Malonyl-CoA is the basic building block for synthesizing a range of important compounds including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metabolism and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomolecular circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metabolism for various biotechnological applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biology strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be critical to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concentration. Applications of malonyl-CoA biosensors as a sensor-invertor for negative feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metabolism and cost-competitively manufacture malonyl-CoA-derived compounds.

Lytic cell death induced by melittin bypasses pyroptosis but induces NLRP3 inflammasome activation and IL-1ß release.

The nucleotide-binding domain and leucine-rich repeat-containing receptor with a pyrin domain 3 (NLRP3) inflammasome is a sensor for different types of infections and alterations of homeostatic parameters, including abnormally high levels of the extracellular nucleotide ATP or crystallization of different metabolites. All NLRP3 activators trigger a similar intracellular pathway, where a decrease in intracellular K+ concentration and permeabilization of plasma membrane are key steps. Cationic amphipathic antimicrobial peptides and peptide toxins permeabilize the plasma membrane. In fact, some of them have been described to activate the NLRP3 inflammasome. Among them, the bee venom antimicrobial toxin peptide melittin is known to elicit an inflammatory reaction via the NLRP3 inflammasome in response to bee venom. Our study found that melittin induces canonical NLRP3 inflammasome activation by plasma membrane permeabilization and a reduction in the intracellular K+ concentration. Following melittin treatment, the apoptosis-associated speck-like protein, an adaptor protein with a caspase recruitment domain (ASC), was necessary to activate caspase-1 and induce IL-1ß release. However, cell death induced by melittin prevented the formation of large ASC aggregates, amplification of caspase-1 activation, IL-18 release and execution of pyroptosis. Therefore, melittin-induced activation of the NLRP3 inflammasome results in an attenuated inflammasome response that does not result in caspase-1 dependent cell death.

An Efficient Transformation Method for the Bioplastic-Producing "Knallgas" Bacterium Ralstonia eutropha H16.

Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a Gram-negative lithoautotrophic ß-proteobacterium with increasing biotechnological applications, including carbon capture and utilization, biopolymer synthesis, and biofuel production. Engineering of this organism is supported by the availability of its genome sequence and suitable plasmid systems. However, the lack of a simple and robust transformation method remains a challenge as it limits both the pace and ease of engineering this organism. To overcome this limitation, a systematic study is performed to evaluate the effects of different parameters on the transformation efficiency of R. eutropha H16. The optimized electroporation protocol uses R. eutropha H16 cells grown to OD600 0.6. These cells are made competent by a 15-min incubation in 50 mM CaCl2 , followed by two cell washes and final resuspension in 0.2 M sucrose prior to electroporation using 2.3 kV. This protocol achieves a transformation efficiency of (3.86 ± 0.29) × 105 cfu µg-1 DNA, a 103 -fold improvement compared to a previously published value for the same plasmid. This transformation method is a valuable tool for R. eutropha H16 research and will further enable the development of other advanced molecular biology methods for this industrially relevant microorganism.

Calprotectina fecal, marcador eficaz en la diferenciación de enfermedades inflamatorias intestinales y trastornos funcionales gastrointestinales / Faecal calprotectin, an useful marker in discriminating between inflammatory bowel disease and functional gastrointestinal disorders

INTRODUCCIÓN: La discriminación entre enfermedades inflamatorias intestinales (EII) y trastornos funcionales gastrointestinales es compleja debido a que pueden presentar cuadros clínicos similares. La calprotectina fecal (CPF) es un marcador inflamatorio útil para este fin y permite seleccionar a los pacientes que más se pueden beneficiar de someterse a una colonoscopia. El objetivo fundamental de este estudio fue valorar la utilidad de la CPF para diferenciar entre enfermedades orgánicas y funcionales. MATERIAL Y MÉTODOS: Se determinó la concentración de CPF de 264 pacientes que presentaban signos o síntomas gastrointestinales indicativos de enfermedad orgánica, y se calculó su precisión diagnóstica mediante el área bajo la curva a partir del diagnóstico final. RESULTADOS: Los pacientes con enfermedad orgánica presentaron valores de CPF mayores (mediana 254 μg/g; intervalo de confianza [IC] 95%, rango intercuartil 105-588,5) que el grupo con enfermedad funcional (95 μg/g; IC 95%, 47,25-243,92; p < 0,0001), así como el grupo con EII (270,85 μg/g; IC 95%, 96,85-674,00) obtuvo valores más elevados que el grupo con síndrome del intestino irritable (79,70; IC 95%, 36,50-117,25; p < 0,0001). Para un valor de corte de 150 μg/g se obtuvo un valor del área bajo la curva de 0,718 para discriminar entre enfermedad orgánica y funcional, y de 0,872 para discriminar entre síndrome del intestino irritable y EII. CONCLUSIÓN: En este estudio se corrobora el alto valor de la CPF en la evaluación de pacientes con sospecha de EII. La mejor eficacia diagnóstica se consigue con un cut-off de 150μg/g para la discriminación entre EII y síndrome del intestino irritable

INTRODUCTION: Diagnostic discrimination between inflammatory bowel disease (IBD) and functional gastrointestinal disorders is complex, as they cause similar signs and symptoms. Faecal calprotectin (FC) is a useful marker in this context, and can be used to select patients who will most benefit from colonoscopy. The aim of this study was to evaluate the utility of FC in discriminating between organic disease and functional disorders. MATERIAL AND METHODS: The study included 264 patients presenting with gastrointestinal complaints consistent with an organic pathology. FC levels were determined and diagnostic accuracy was assessed using the area under the curve obtained from the final diagnosis. RESULTS: Calprotectin levels in organic bowel disease patients were significantly higher (median 254 μ g/g; 95% confidence interval [CI], interquartile range 105-588.5) than in functional disease patients (95 μ g/g; 95% CI, 47.25-243.92) (P < .0001). Similarly, in patients with IBD, the values obtained were higher (270.85 μg/g; 95% CI, 96.85-674.00) than in those with irritable bowel syndrome (79.70μ g/g; 95% CI, 36.50-117.25) (P<.0001). For a cut-off of 150 μ g/g, FC had an area under the ROC curve to discriminate between organic and functional disease of 0.718, and 0.872 to discriminate between irritable bowel syndrome and IBD. CONCLUSION: Our study supports the importance of FC as a marker in the evaluation of patients with IBD. The best diagnostic accuracy is obtained at a cut-off value of 150μ g/g

Faecal calprotectin, an useful marker in discriminating between inflammatory bowel disease and functional gastrointestinal disorders. / Calprotectina fecal, marcador eficaz en la diferenciación de enfermedades inflamatorias intestinales y trastornos funcionales gastrointestinales.

INTRODUCTION: Diagnostic discrimination between inflammatory bowel disease (IBD) and functional gastrointestinal disorders is complex, as they cause similar signs and symptoms. Faecal calprotectin (FC) is a useful marker in this context, and can be used to select patients who will most benefit from colonoscopy. The aim of this study was to evaluate the utility of FC in discriminating between organic disease and functional disorders. MATERIAL AND METHODS: The study included 264 patients presenting with gastrointestinal complaints consistent with an organic pathology. FC levels were determined and diagnostic accuracy was assessed using the area under the curve obtained from the final diagnosis. RESULTS: Calprotectin levels in organic bowel disease patients were significantly higher (median 254µg/g; 95% confidence interval [CI], interquartile range 105-588.5) than in functional disease patients (95µg/g; 95% CI, 47.25-243.92) (P<.0001). Similarly, in patients with IBD, the values obtained were higher (270.85µg/g; 95% CI, 96.85-674.00) than in those with irritable bowel syndrome (79.70µg/g; 95% CI, 36.50-117.25) (P<.0001). For a cut-off of 150µg/g, FC had an area under the ROC curve to discriminate between organic and functional disease of 0.718, and 0.872 to discriminate between irritable bowel syndrome and IBD. CONCLUSION: Our study supports the importance of FC as a marker in the evaluation of patients with IBD. The best diagnostic accuracy is obtained at a cut-off value of 150µg/g.

The COL1A1 SP1 polymorphism is associated with lower bone mineral density in patients treated with valproic acid.

OBJECTIVE: The aim of this study was to determine the possible relationship between the Sp1 polymorphism of gene COL1A1 and bone metabolism disorder in individuals with epilepsy. METHODS: To this end, we carried out an observational cross-sectional study on 64 patients in monotherapy with an antiepileptic drug. The patients were classified on the basis of the presence of the 's' allele of the COL1A1 Sp1 polymorphism. RESULTS: In the patients with SS, the standardized bone mineral density (sBMD) in the left femoral neck was 1024.9±206.1 mg/cm, whereas in the patients with Ss or ss, the density was significantly lower, 917±141.4 mg/cm (P=0.027), as was the femoral t-score (0.72±1.67 vs. -0.29±1.15, P=0.01). The values in the lumbar spine were equally greater in those with SS: 1219.1±236.3 versus 1090.5±142.7 mg/cm for the sBMD (P=0.018) and 0.67±1.98 versus -0.34±1.16 for the lumbar t-score (P=0.023). The bone biomarkers showed no significant differences nor did the 25-OH vitamin D and parathormone values. In the patient group treated with valproic acid (VPA), the densitometric values were significantly lower in the Ss or ss patients compared with SS homozygotes: 887.1±142.6 versus 1120.6±198.2 mg/cm for femoral sBMD (P=0.02), 990±98.1 versus 1417±251.2 mg/cm for lumbar sBMD (P=0.001). Of the patients who were carriers of the 's' allele and who were treated with VPA, 86% achieved osteopenia values. CONCLUSION: In our study, the presence of the 's' allele of the COL1A1 Sp1 polymorphism in individuals with epilepsy was related to lower bone BMD (lumbar and femoral). This relationship seemed to be further apparent in the patients undergoing treatment with VPA.