SecMet-FISH: Labeling, visualization, and enumeration of secondary metabolite producing microorganisms

Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra, which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community

Assessment of preoperative and postoperative prealbumin in thoracic surgery – a two months experience in a Romanian university hospital / Evaluarea preoperatorie şi postoperatorie a prealbuminei în chirurgia toracică - experiența de 2 luni a unui spital universitar din România

Malnutriția este o constatare frecventă și importantă în secțiile chirurgicale. Deşi consecințele sale includ complicații postoperatorii și costuri mai mari, evaluarea nutrițională nu face parte din protocoalele preoperatorii de rutină. Evaluarea nutriţională include parametri clinici și biologici și este vitală pentru a începe tratamentul și a îmbunătăți rezultatele. Prealbumina este în prezent recunoscută ca un marker fidel al malnutriției şi este inclusă în ghidurile de practică. Unul dintre cele mai importante aspecte legate de prealbumină este faptul că variațiile sale în timp sunt mai valoroase decât valorile absolute. Scopul acestui studiu a fost de a evalua și compara evoluția nutrițională perioperatorie a pacienţilor care necesită intervenţii de chirurgie toracică, cu şi fără cancer, folosind prealbumina - preoperator și postoperator - ca marker principal. Treizeci şi şase de pacienţi de la Clinica de Chirurgie Toracică au fost evaluaţi înainte de intervenţia chirurgicală, prin indicele de masă corporală, scorul de risc nutriţional Subjective Global Assessment (SGA) și parametri biochimici de rutină. Prealbumina a fost evaluată înainte de intervenţia chirurgicală şi la 3 zile după operaţie.Vârstă, durata spitalizării postoperatorii și prezența complicaţiilor au fost notate. Pacienții cu cancer (n = 19) au fost semnificativ mai în vârstă decât pacienții fără cancer (p = 0,007) și au fost mai frecvent, dar nu în mod semnificativ, evaluaţi prin SGA ca fiind malnutriţi (42,1% față de 11,6%). Prealbumina preoperator și alți parametri nu diferă semnificativ între grupuri. Cu toate acestea, a existat o scădere semnificativă a prealbuminei postoperator doar la pacienții cu cancer. Prin urmare, prealbumina s-a dovedit a fi valoroasă în evaluarea malnutriţiei acute la pacienţii cu cancer, în special prin monitorizarea variaţiilor în timp, ceea ce ar putea fi util în planificarea tratamentului nutriţional

Bacterial Biodegradation of Perfluorooctanoic Acid (PFOA) and Perfluorosulfonic Acid (PFOS) Using Pure <i>Pseudomonas</i> Strains

The principal objective of the present research involved the achievement of high biodegradation degrees of perfluorooctanoic acid (PFOA) and perfluorosulfonic acid (PFOS) using pure individual bacterial strains. The use of such microorganisms can contribute to the improvement of the wastewater treatment process in sewage treatment plants through bioaugmentation or other bioremediation processes. Thus, in this study, we investigated the biodegradation potential of PFOA and PFOS. Bacterial strains tested in this study were from the Pseudomonas genus, namely: Pseudomonas aeruginosa and Pseudomonas putida, due to their known capacity to degrade xenobiotic compounds. The results indicated that Pseudomonas aeruginosa was able to transform 27.9% of PFOA and 47.3% of PFOS in 96 h, while Pseudomonas putida managed to transform 19.0% of PFOA and 46.9% of PFOS in the same time frame. During the biodegradation tests, PFHxA was recognized as the principal biotransformation product of PFOA in the presence of Pseudomonas aeruginosa, and PFPeA, PFPxA and PFHpA were recognized as the biotransformation products in the presence of Pseudomonas putida. For PFOS, only two biotransformation products (PHHxA and PFHpA) were observed as a consequence of biodegradation by both bacterial strains

Correlations between PNPLA3 Gene Polymorphisms and NAFLD in Type 2 Diabetic Patients

Background and Objectives: Non-alcoholic fatty liver disease is a worldwide significant public health problem, particularly in patients with type 2 diabetes mellitus. Identifying possible risk factors for the disease is mandatory for a better understandingand management of this condition. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) has been linked to the development and evolution of fatty liver but not to insulin resistance. The aim of this study isto evaluate the relationships between PNPLA3 and fatty liver, metabolic syndrome and subclinical atherosclerosis. Materials and Methods: The study group consisted of patients with type 2 diabetes mellitus without insulin treatment. The degree of liver fat loading was assessed by ultrasonography, and subclinical atherosclerosis was assessed using carotid intima-media thickness (CIMT). PNPLA3 rs738409 genotype determination was performed by high-resolution melting analysis that allowed three standard genotypes: CC, CG, and GG. Results: Among the 92 patients, more than 90% showed various degrees of hepatic steatosis, almost 62% presented values over the normal limit for the CIMT. The majority of the included subjects met the criteria for metabolic syndrome. Genotyping of PNPLA3 in 68 patients showed that the difference between subjects without steatosis and subjects with hepatic steatosis was due to the higher frequency of genotype GG. The CC genotype was the most common in the group we studied and was significantly more frequent in the group of subjects with severe steatosis; the GG genotype was significantly more frequent in subjects with moderate steatosis; the frequency of the CG genotype was not significantly different among the groups.When we divided the group of subjects into two groups: those with no or mild steatosis and those with moderate or severe steatosis it was shown that the frequency of the GG genotype was significantly higher in the group of subjects with moderate or severe steatosis. PNPLA3 genotypes were not associated with metabolic syndrome, subclinical atherosclerosis, or insulin resistance. Conclusions: Our results suggest that PNPLA3 does not independently influence cardiovascular risk in patients with type 2 diabetes mellitus. The hypothesis that PNPLA3 may have a cardioprotective effect requires future confirmation