Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Introduction: Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. Methods: We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring ß-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Results: Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 µm observed at 5 days. Functionality, assessed through ß-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. Discussion: This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

Simultaneous detection of arboviruses by a multiplex RT-qPCR assay in Tocantins, a northern state of Brazil.

In Brazil, Dengue, Zika and Chikungunya viruses constitute a major threat to the public health system. Simultaneous circulation of these arboviruses occurs in many regions of the world due to the expansion of transmission vectors. The infection by these arboviruses triggers similar symptoms during their acute phase. However, in some cases, severe symptoms may occur, leading to different types of disabilities and even death. In this context, considering the similarity of the symptoms, the problems caused by the infection of these arboviruses, and the increasing risk of coinfection in humans, the differential diagnosis of these infections is essential for clinical management and epidemiological investigation. Thus, this study aimed to identify, through diagnosis via Quantitative Polymerase Chain Reaction with Reverse Transcription, arbovirus coinfection in patients from the Tocantins state (Northern Brazil). A total of 495 samples were analyzed, three from which were determined to be a coinfection of Dengue and Chikungunya viruses. The data obtained here indicate the co-circulation and coinfection by Dengue and Chikungunya viruses in the Tocantins state. These results highlight the importance of monitoring the circulation of these arboviruses for the development of health actions that aim their prevention and combat, as well as their clinical and therapeutic management.

Multiple antibiotic resistance and herbicide catabolic profiles of bacteria isolated from Lake Villarrica surface sediments (Chile).

Antibiotics and herbicides are contaminants of emerging concern in aquatic environments. Lake Villarrica is a relevant freshwater body in Chile and was recently designated a 'saturated nutrient zone'. Here, we investigated the occurrence of multiple antibiotic resistance (MAR) and herbicide catabolic profiles among bacteria present in the surface sediments of Lake Villarrica. The occurrence of antibiotic-resistant genes (ARGs; blaTEM, catA and tetM) and herbicide-catabolic genes (HCGs; phnJ and atzA) was investigated by qPCR. Subsequently, the presence of culturable bacteria with multiple resistance to amoxicillin (AMX), chloramphenicol (CHL) and oxytetracycline (OXT) was studied. Forty-six culturable MAR (AMX + CHL + OXT) strains were isolated and characterized with respect to their resistance to 11 antibiotics by using a disc diffusion assay and testing their ability to use herbicides as a nutrient source. qPCR analyses revealed that ARGs and HCGs were present in all sediment samples (101 to 103 gene copies g-1), with significant (P ≤ 0.05) higher values in sites near Villarrica city and cattle pastures. The plate method was used to recover MAR isolates from sediment (103-106 CFU g-1), and most of the 46 isolates also showed resistance to oxacillin (100%), cefotaxime (83%), erythromycin (96%) and vancomycin (93%). Additionally, 54 and 57% of the MAR isolates were able to grow on agar supplemented (50 mg L-1) with atrazine and glyphosate as nutrient sources, respectively. Most of the MAR isolates were taxonomically close to Pseudomonas (76.1%) and Pantoea (17.4%), particularly those isolated from urbanized sites (Pucón city). This study shows the presence of MAR bacteria with herbicide catabolic activity in sediments, which is valuable for conservation strategies and risk assessments of Lake Villarrica. However, major integrative studies on sediments as reservoirs or on the fate of MAR strains and traces of antibiotics and herbicides as a result of anthropic pressure are still needed.

Development of a TaqMan Assay for the Detection of 'Candidatus Phytoplasma brasiliense' and Assessment by High-Resolution Melt Curve Analysis.

'Candidatus Phytoplasma brasiliense' (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp., where it causes witches broom symptoms, and papaya, where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study, two quantitative PCR assays are developed: one using high-resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMan assay successfully amplified two of the three isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII, and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting melting point (Tm) products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.

Spread and persistence of antimicrobial resistance genes in wastewater from human and animal sources in São Paulo, Brazil.

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (blaCTX-M, blaKPC, qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes blaCTX-M, qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems.

Simultaneous detection of arboviruses by a multiplex RT-qPCR assay in Tocantins, a northern state of Brazil

ABSTRACT In Brazil, Dengue, Zika and Chikungunya viruses constitute a major threat to the public health system. Simultaneous circulation of these arboviruses occurs in many regions of the world due to the expansion of transmission vectors. The infection by these arboviruses triggers similar symptoms during their acute phase. However, in some cases, severe symptoms may occur, leading to different types of disabilities and even death. In this context, considering the similarity of the symptoms, the problems caused by the infection of these arboviruses, and the increasing risk of coinfection in humans, the differential diagnosis of these infections is essential for clinical management and epidemiological investigation. Thus, this study aimed to identify, through diagnosis via Quantitative Polymerase Chain Reaction with Reverse Transcription, arbovirus coinfection in patients from the Tocantins state (Northern Brazil). A total of 495 samples were analyzed, three from which were determined to be a coinfection of Dengue and Chikungunya viruses. The data obtained here indicate the co-circulation and coinfection by Dengue and Chikungunya viruses in the Tocantins state. These results highlight the importance of monitoring the circulation of these arboviruses for the development of health actions that aim their prevention and combat, as well as their clinical and therapeutic management.

Multiple myeloma and Chagas disease: qPCR as a marker for preemptive antiparasitic therapy: a case reports series and review

ABSTRACT Multiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56±32.10 months (mean±SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR.

Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia.

Visceral leishmaniasis (VL) is a neglected disease considered a serious public health problem, especially in endemic countries. Several studies have discovered monoxenous trypanosomatids (Leptomonas and Crithidia) in patients with VL. In different situations of leishmaniasis, investigations have examined cases of co-infection between Leishmania spp. and Crithidia spp. These coinfections have been observed in a wide range of vertebrate hosts, indicating that they are not rare. Diagnostic techniques require improvements and more robust tools to accurately detect the causative agent of VL. This study aimed to develop a real-time quantitative dye-based PCR (qPCR) assay capable of distinguishing Leishmania infantum from Crithidia-related species and to estimate the parasite load in samples of VL from humans and animals. The primer LinJ31_2420 targets an exclusive phosphatase of L. infantum; the primer Catalase_LVH60-12060_1F targets the catalase gene of Crithidia. Therefore, primers were designed to detect L. infantum and Crithidia sp. LVH60A (a novel trypanosomatid isolated from VL patients in Brazil), in samples related to VL. These primers were considered species-specific, based on sequence analysis using genome data retrieved from the TriTryp database and the genome assembling of Crithidia sp. LVH60A strain, in addition to experimental and clinical data presented herein. This novel qPCR assay was highly accurate in identifying and quantifying L. infantum and Crithidia sp. LVH60A in samples obtained experimentally (in vitro and in vivo) or collected from hosts (humans, dogs, cats, and vectors). Importantly, the screening of 62 cultured isolates from VL patients using these primers surprisingly revealed that 51 parasite cultures were PCR+ for Crithidia sp. In addition, qPCR assays identified the co-infection of L. infantum with Crithidia sp. LVH60A in two new VL cases in Brazil, confirming the suspicion of co-infection in a previously reported case of fatal VL. We believe that the species-specific genes targeted in this study can be helpful for the molecular diagnosis of VL, as well as for elucidating suspected co-infections with monoxenous-like trypanosomatids, which is a neglected fact of a neglected disease.

Mixed infection of Bartonella and Eperythrozoon in a dog: case report / Infecção mista de Bartonella e Eperythrozoon em um cão: relato de caso

A March male Golden, weighing 7.6kg, presented with gradual weight loss, high body temperature, depression, poor appetite and thirst, and vomiting before consultation. The results showed that the erythrocytes, hematocrit, hemoglobin, and platelets were lower than the reference values. The diagnosis of mixed infection with haematocrit and eosinophilic bodies was confirmed by real-time fluorescence PCR of whole blood, which was positive for haematocrit and eosinophilic bodies. The dog was treated with doxycycline and ceftriaxone, and the dog fully recovered after 2 weeks with blood transfusion, symptomatic treatment, and supportive therapy. This indicates that the disease can be treated well by a comprehensive treatment approach.

Um March Golden macho, pesando 7,6kg, apresentou perda de peso gradual, temperatura corporal alta, depressão, falta de apetite e sede, e vômitos antes da consulta. Os resultados mostraram que os eritrócitos, hematócrito, hemoglobina e plaquetas eram menores que os valores de referência. O diagnóstico de infecção mista com hematócrito e corpos eosinófilos foi confirmado por PCR de fluorescência em tempo real de sangue total, o que foi positivo para hematócrito e corpos eosinófilos. O cão foi tratado com doxiciclina e ceftriaxona, e o cão recuperou-se completamente após duas semanas com transfusão de sangue, tratamento sintomático e terapia de suporte. Isto indica que a doença pode ser bem tratada através de uma abordagem de tratamento abrangente.

Differential analysis of culturable and unculturable subgingival target microorganisms according to the stages of periodontitis.

OBJECTIVES: Culturable and unculturable microorganisms have been associated with periodontitis. Their differential proportions and composition have not been evaluated by their severity and complexity defined by stages in the 2018 AAP-EEP classification. METHODS: One hundred eighty subgingival biofilm samples were collected in Spain and Colombia from subjects categorized as health/gingivitis: periodontitis stages I/II periodontitis stages III/IV. Target culturable microorganisms (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Eubacterium nodatum) and target unculturable microorganisms (Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis) were evaluated by quantitative PCR analysis. In addition, their differences and association with periodontal status were analyzed by ANCOVA and logistic regression models once adjusted to age, current smoking, and country. RESULTS: P. gingivalis was significantly associated with periodontitis stages I/II, OR 2.44 (CI 95% 1.08-5.47) and stages III/V, OR 6.43 (CI 95% 2.43-16.9). T forsythia, OR 7.53 (CI 95% 2.07-27.4); D. oralis, OR 5.99 (CI 95% 2.71-13.23); F. alocis, OR 10.9 (CI 95% 4.56-23.2); E. brachy, 3.57 (CI 95% 1.40-9.11); and E. saphenum, 4.85 (CI 95% 1.99-11.7) were significantly associated only with stages III/IV periodontitis. P. gingivalis evidenced significant differences with the increase in the severity of the periodontal lesion: 2.97 colony forming unit (CFU)/µL (CI 95% 2.32-3.54) health/gingivitis, and 4.66 CFU/µL (CI 95% 4.03-5.30) and 5.90 CFU/µL (CI 95% 5.20-6.48) in stages I/II and III/IV respectively (p < 0.0001). Unculturable microorganisms only evidenced differences in concentration in stages III/IV compared with health-gingivitis (p ≤ 0.001). CONCLUSION: Culturable and unculturable are strongly associated with stages III/IV periodontitis. Classic culturable microorganisms are more sensitive to differentiate between stages of periodontitis in the quantitative analysis. CLINICAL RELEVANCE: Future interventional studies of periodontal disease should include Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, and Desulfobulbus oralis as possible markers of therapy response and as indicators of progressive disease.

Molecular diagnosis of opportunistic infections in the central nervous system of HIV-infected adults in Manaus, Amazonas.

Background: Opportunistic infections in the central nervous system (CNS) of people with HIV/AIDS (PLWHA) remain significant contributors to morbidity and mortality, especially in resource-limited scenarios. Diagnosing these infections can be challenging, as brain imaging is non-specific and expensive. Therefore, molecular analysis of cerebrospinal fluid (CSF) may offer a more accurate and affordable method for diagnosing pathogens. Methods: We conducted extensive real-time PCR testing (qPCR) on CSF to evaluate etiological agents in PLWHA with neurological manifestations. Primers targeting DNA from specific pathogens, including cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), John Cunningham virus (JCV), Toxoplasma gondii, and human T-lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2), were used. Results: Cerebrospinal fluid samples revealed 90 pathogens (36.7%). Toxoplasma gondii was the most frequently detected pathogen, found in 22 samples (30.5%). Other pathogens included Cryptococcus sp. (7.7%), EBV (5.3%), CMV, VZV, and JCV (4.0% each). Conclusion: Despite antiretroviral therapy and medical follow-up, opportunistic central nervous system infections remain frequent in PLWHA. Herpesviruses are commonly detected, but T. gondii is the most prevalent opportunistic pathogen in our study population. Therefore, molecular diagnosis is a crucial tool for identifying opportunistic infections, even in patients undergoing treatment.

Quantitative Detection of Leishmania in Rhipicephalus microplus and Amblyomma sabanerae in the Peruvian Amazon Basin.

Leishmaniasis is a disease of public importance with a complex transmission cycle. A quantitative PCR was developed by using the small subunit of the ribosomal RNA gene (SSU rRNA) as a DNA target, which is conserved in all Leishmania species. A TaqMan ® probe was designed to have a high specificity. In all, 22 out of 23 (95.7%) ticks classified as R. microplus tested positive for Leishmania sp. The quantification was between 34.1 and 2197.1 parasites per tick in a range of 12 to 769 fg/uL. In addition, 9 out of 10 (90%) ticks classified as Amblyomma sabanerae tested positive for Leishmania sp. The quantification was between 448.6 and 5428.6 parasites per tick in a range of 157 to 1900 fg/µL. Leishmania sp. was identified in very high percentages in Rhipicephalus microplus and Amblyomma sabanerae from wild Pecari tajacu and Chelonoidis denticulata, in quantities of 34.1 and 5428.6 parasites per arthropod, and this could suggest that the ticks were parasitized by sucking blood from the animals from which they were collected. This is the first report about Leishmania parasites found in wild Rhipicephalus microplus and Amblyomma sabanerae, adding new information about the distribution and epidemiology of the parasite in sylvatic areas.

Inoculation of Herbaspirillum seropedicae strain SmR1 increases biomass in maize roots DKB 390 variety in the early stages of plant development.

Herbaspirillum seropedicae is a plant growth-promoting bacteria isolated from diverse plant species. In this work, the main objective was to investigate the efficiency of H. seropedicae strain SmR1 in colonizing and increasing maize growth (DKB 390 variety) in the early stages of development under greenhouse conditions. Inoculation with H. seropedicae resulted in 19.43 % (regarding High and Low N controls) and 10.51% (regarding Low N control) in mean of increase of root biomass, for 1st and 2nd greenhouse experiments, respectively, mainly in the initial stages of plant development, at 21 days after emergence (DAE). Quantification of H. seropedicae in roots and leaves was performed by quantitative PCR. H. seropedicae was detected only in maize inoculated roots by qPCR, and a slight decrease in DNA copy number g-1 of fresh root weight was observed from 7 to 21 DAE, suggesting that there was initial effective colonization on maize plants. H. seropedicae strain SmR1 efficiently increased maize root biomass exhibiting its potential to be used as inoculant in agricultures systems.

Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection.

Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/µL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV.

Diversity of Microbial Functional Genes Should Be Considered During the Interpretation of the qPCR Melting Curves.

Soil microorganisms play an essential role in biogeochemical cycles. One approach to study these microbial communities is quantifying functional genes by quantitative PCR (qPCR), in which a melting curve analysis is usually assessed to confirm that a single PCR product is being quantified. However, the high diversity of functional genes in environmental samples could generate more than one peak in those curves, so the presence of two or multiple peaks does not always indicate nonspecific amplification. Here, we analyzed the taxonomic diversity of soil microorganisms harboring functional genes involved in nitrogen (N) and phosphorus (P) cycles, based on a database of genomes and metagenomes, and predicted the melting curve profiles of these genes. These functional genes were spread across many bacterial phyla, but mainly Proteobacteria and Actinobacteria. In general, the melting curves exhibited more than one peak or peaks with shoulders, mainly related to the variation of the nucleotide composition of the genes and the expected size of the amplicons. These results indicate that the melting curves of functional genes from environmental samples should be carefully evaluated, being in silico analyses a cost-effective way to identify inherent sequence diversity and avoid interpreting multiple peaks always as unspecific amplifications.

Knowing the enemy: homoacetogens in hydrogen production reactors.

One of the bottlenecks of the hydrogen production by dark fermentation is the low yields obtained because of the homoacetogenesis persistence, a metabolic pathway where H2 and CO2 are consumed to produce acetate. The central reactions of H2 production and homoacetogenesis are catalyzed by enzyme hydrogenase and the formyltetrahydrofolate synthetase, respectively. In this work, genes encoding for the formyltetrahydrofolate synthetase (fthfs) and hydrogenase (hydA) were used to investigate the diversity of homoacetogens as well as their phylogenetic relationships through quantitative PCR (qPCR) and next-generation amplicon sequencing. A total of 70 samples from 19 different H2-producing bioreactors with different configurations and operating conditions were analyzed. Quantification through qPCR showed that the abundance of fthfs and hydA was strongly associated with the type of substrate, organic loading rate, and H2 production performance. In particular, fthfs sequencing revealed that homoacetogens diversity was low with one or two dominant homoacetogens in each sample. Clostridium carboxivorans was detected in the reactors fed with agave hydrolisates; Acetobacterium woodii dominated in systems fed with glucose; Blautia coccoides and unclassified Sporoanaerobacter species were present in reactors fed with cheese whey; finally, Eubacterium limosum and Selenomonas sp. were co-dominant in reactors fed with glycerol. Altogether, quantification and sequencing analysis revealed that the occurrence of homoacetogenesis could take place due to (1) metabolic changes of H2-producing bacteria towards homoacetogenesis or (2) the displacement of H2-producing bacteria by homoacetogens. Overall, it was demonstrated that the fthfs gene was a suitable marker to investigate homoacetogens in H2-producing reactors. KEY POINTS: • qPCR and sequencing analysis revealed two homoacetogenesis phenomena. • fthfs gene was a suitable marker to investigate homoacetogens in H2 reactors.

Validation and Selection of New Reference Genes for RT-qPCR Analysis in Pediatric Glioma of Different Grades.

Gliomas are heterogeneous, solid, and intracranial tumors that originate from glial cells. Malignant cells from the tumor undergo metabolic alterations to obtain the energy required for proliferation and the invasion of the cerebral parenchyma. The alterations in the expression of the genes related to the metabolic pathways can be detected in biopsies of gliomas of different CNS WHO grades. In this study, we evaluated the expression of 16 candidate reference genes in the HMC3 microglia cell line. Then, statistical algorithms such as BestKeeper, the comparative ΔCT method, geNorm, NormFinder, and RefFinder were applied to obtain the genes most suitable to be considered as references for measuring the levels of expression in glioma samples. The results show that PKM and TPI1 are two novel genes suitable for genic expression studies on gliomas. Finally, we analyzed the expression of genes involved in metabolic pathways in clinical samples of brain gliomas of different CNS WHO grades. RT-qPCR analysis showed that in CNS WHO grade 3 and 4 gliomas, the expression levels of HK1, PFKM, GAPDH, G6PD, PGD1, IDH1, FASN, ACACA, and ELOVL2 were higher than those of CNS WHO grade 1 and 2 glioma biopsies. Hence, our results suggest that reference genes from metabolic pathways have different expression profiles depending on the stratification of gliomas and constitute a potential model for studying the development of this type of tumor and the search for molecular targets to treat gliomas.

Climatological and Epidemiological Conditions Are Important Factors Related to the Abundance of blaKPC and Other Antibiotic Resistance Genes (ARGs) in Wastewater Treatment Plants and Their Effluents, in an Endemic Country.

Several physicochemical and season factors have been related to the abundance of antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs), considered hotspots of bacterial resistance. However, few studies on the subject have been carried out in tropical countries endemic for resistance mechanisms such as blaKPC. In this study, the occurrence of ARGs, particularly blaKPC, was determined throughout a WWTP, and the factors related to their abundance were explored. In 2017, wastewater samples were taken from a WWTP in Colombia every 15 days for 6 months, and a total of 44 samples were analyzed by quantitative real-time PCR. sul1, sul2, blaKPC, and ermB were found to be the most prevalent ARGs. A low average reduction of the absolute abundance ARGs in effluent with respect to influent was observed, as well as a greater absolute abundance of ARGs in the WWTP effluent in the rainy season. Factors such as temperature, pH, oxygen, total organic carbon (TOC), chemical oxygen demand (COD), and precipitation were significantly correlated with the absolute abundance of several of the ARGs evaluated. A generalized linear mixed-effects model analysis showed that dissolved oxygen and precipitation in the sampling day were important factors related to the absolute concentration of blaKPC over time. In conclusion, the abundance of ARGs in the WWTP could be influenced by endemic conditions and physicochemical and climatological parameters. Therefore, it is necessary to continuously monitor clinical relevant genes in WWTPs from different global regions, even more so in low-income countries where sewage treatment is limited.

Differential Expression of Myogenic and Calcium Signaling-Related Genes in Broilers Affected With White Striping.

White Striping (WS) has been one of the main issues in poultry production in the last years since it affects meat quality. Studies have been conducted to understand WS and other myopathies in chickens, and some biological pathways have been associated to the prevalence of these conditions, such as extracellular calcium level, oxidative stress, localized hypoxia, possible fiber-type switching, and cellular repairing. Therefore, to understand the genetic mechanisms involved in WS, 15 functional candidate genes were chosen to be analyzed by quantitative PCR (qPCR) in breast muscle of normal and WS-affected chickens. To this, the pectoral major muscle (PMM) of 16 normal and 16 WS-affected broilers were collected at 42 days of age and submitted to qRT-PCR analysis. Out of the 15 genes studied, six were differentially expressed between groups. The CA2, CSRP3, and PLIN1 were upregulated, while CALM2, DNASE1L3, and MYLK2 genes were downregulated in the WS-affected when compared to the normal broilers. These findings highlight that the disruption on muscle and calcium signaling pathways can possibly be triggering WS in chickens. Improving our understanding on the genetic basis involved with this myopathy might contribute for reducing WS in poultry production.

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts.

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.