Classification and counting of cells in brightfield microscopy images: an application of convolutional neural networks.

Microscopy is integral to medical research, facilitating the exploration of various biological questions, notably cell quantification. However, this process's time-consuming and error-prone nature, attributed to human intervention or automated methods usually applied to fluorescent images, presents challenges. In response, machine learning algorithms have been integrated into microscopy, automating tasks and constructing predictive models from vast datasets. These models adeptly learn representations for object detection, image segmentation, and target classification. An advantageous strategy involves utilizing unstained images, preserving cell integrity and enabling morphology-based classification-something hindered when fluorescent markers are used. The aim is to introduce a model proficient in classifying distinct cell lineages in digital contrast microscopy images. Additionally, the goal is to create a predictive model identifying lineage and determining optimal quantification of cell numbers. Employing a CNN machine learning algorithm, a classification model predicting cellular lineage achieved a remarkable accuracy of 93%, with ROC curve results nearing 1.0, showcasing robust performance. However, some lineages, namely SH-SY5Y (78%), HUH7_mayv (85%), and A549 (88%), exhibited slightly lower accuracies. These outcomes not only underscore the model's quality but also emphasize CNNs' potential in addressing the inherent complexities of microscopic images.

Cell quantification in digital contrast microscopy images with convolutional neural networks algorithm.

High Content Screening (HCS) combines high throughput techniques with the ability to generate cellular images of biological systems. The objective of this work is to evaluate the performance of predictive models using CNN to identify the number of cells present in digital contrast microscopy images obtained by HCS. One way to evaluate the algorithm was through the Mean Squared Error metric. The MSE was 4,335.99 in the A549 cell line, 25,295.23 in the Huh7 and 36,897.03 in the 3T3. After obtaining these values, different parameters of the models were changed to verify how they behave. By reducing the number of images, the MSE increased considerably, with the A549 cell line changing to 49,973.52, Huh7 to 79,473.88 and 3T3 to 52,977.05. Correlation analyzes were performed for the different models. In lineage A549, the best model showed a positive correlation with R = 0.953. In Huh7, the best correlation of the model was R = 0.821, it was also a positive correlation. In 3T3, the models showed no correlation, with the best model having R = 0.100. The models performed well in quantifying the number of cells, and the number and quality of the images interfered with this predictive ability.

Controlling antimicrobial activity and drug loading capacity of chitosan-based layer-by-layer films.

We report on layer-by-layer (LbL) films of chitosans (CHI) and hyaluronic acid (HA) whose properties could be controlled by employing chitosans with different degrees of deacetylation (DD¯ ≈ 85%; 65%; 40%) and high average molecular weight (ca. 106 g/mol). In spite of their high molecular weight, these chitosans are soluble within a wide pH range, including physiological pH. HA/CHI LbL films produced from polymer solutions at pH 4.5 were thinner, smoother, more hydrophilic than those prepared at pH 7.2. This is attributed to the more extended conformation adopted by chitosan due to its very high charge density at low pH, favoring a compact chain packing during the film formation and resulting in lower film thickness and roughness. The smoother HA/CHI LbL films obtained at pH 4.5 were effective against Escherichia coli, while the thicker, rougher LbL films fabricated at pH 7.2 could be used in the controlled released of Rose Bengal dye. In summary, the tuning of only two parameters, i.e. solution pH and DD¯ of chitosans, provides access to a library of HA/CHI LbL films for tailored, diversified applications.

Effect of fragmentation on the natural genetic diversity of Theobroma speciosum Willd. ex Spreng. populations.

Forest fragmentation reduces the effective size of natural populations, isolates individuals in the landscape, and, consequently, changes species' mating systems by increasing the degree of relatedness between individuals and inbreeding. Investigating the impact of habitat degradation on forest fragments helps to assess the genetic and ecological consequences of these changes, and allows the development of effective and sustainable conservation strategies to manage the genetic resources of species living in degraded landscapes. The aim of the present study was to assess the genetic diversity of fragmented Theobroma speciosum populations using microsatellite markers. Three urban forest fragments were selected in the municipality of Alta Floresta, Mato Grosso State, Brazil, namely C/E park, J park, and Zoo Botanical park. Seventy-five individuals (25 in each fragment) were sampled by collecting their leaves for genomic DNA extraction. Polymerase chain reaction amplifications were performed using nine polymorphic simple sequence repeat primers, which amplified 84 alleles. The mean expected heterozygosity was 0.970, and it was always higher than the observed heterozygosity. Analysis of molecular variance revealed that most variability occurred within populations (64%) rather than between them (36%). The Structure software and an unweighted pair group method with arithmetic mean dendrogram revealed three distinct groups, showing that individuals were allocated to their correct populations. Genotype number 3 from C/E park, number 45 from J park, and number 51 from Zoo Botanical park could be used as stock plants in breeding programs, because they were the most dissimilar within the populations studied. The high genetic diversity levels detected in all three populations studied emphasize the importance of protecting this species in its natural habitat.

Recovery of Arapaima sp. populations by community-based management in floodplains of the Purus River, Amazon.

In the present study a unique dataset on population abundance in various community-based management (CBM) and non-CBM areas is analysed to address the question of whether CBM can recover overexploited populations of Arapaima sp. in river-floodplain ecosystems. All non-CBM areas possessed depleted Arapaima sp. populations with a mean density of 0·01 individuals ha(-1) . Arapaima sp. population densities in all CBM areas changed over time from depleted to overexploited or well managed status, with a mean rate of increase of 77% year(-1) . Rates of Arapaima sp. population recovery in CBM areas differed, probably reflecting differences in ecosystem productivity and compliance with management regulations. These results indicate that CBM schemes can be effective tools for the recovery and conservation of fish populations with non-migratory life cycles in tropical river-floodplain ecosystems.

The effect of leptin concentrations and other maternal characteristics on gestational weight gain is different according to pre-gestational BMI: results from a prospective cohort.

OBJECTIVE: To evaluate the effect of leptin and other selected variables on gestational weight gain (GWG) according to pre-gestational body mass index (BMI). DESIGN: Prospective cohort. SETTING: Public Health Center, Rio de Janeiro, Brazil. SAMPLE: Two hundred and twenty-eight pregnant women. METHODS: Women were followed at the 5-13, 20-26 and 30-36th gestational weeks. The effects of independent variables on GWG in normal weight (BMI 18.5-24.9 kg/m(2) ), overweight (BMI 25.0-29.9 kg/m(2) ) and obese (BMI ≥ 30.0 kg/m(2) ) women were assessed using longitudinal linear mixed-effects models. MAIN OUTCOME MEASURE: Maternal body weight (kg) throughout pregnancy. RESULTS: Leptin concentrations were associated with GWG in normal weight (ß = 0.048, P < 0.001) and overweight (ß = 0.023, P = 0.048) women, but not in obese ones (ß = 0.011, P = 0.308). Additionally, the number of hours slept per night decreased the effect of leptin on GWG in OW women (ß = -0.013, P = 0.002). The effect of other maternal characteristics on GWG was different depending on the BMI category. CONCLUSIONS: Leptin concentrations were positively associated with GWG in normal weight and overweight women, but not in obese ones. Maternal height was associated with GWG in all BMI categories, but other variables such as sleep duration, QUICKI values, HDL-c, smoking habit and marital status presented differential effects according to BMI. We encourage further studies to investigate the association between leptin and gestational weight gain, taking into account the pre-pregnancy weight and sleep duration, in order to compare and confirm our results. TWEETABLE ABSTRACT: Leptin is associated with weight gain in normal weight and overweight pregnant women, but not in obese ones.

Single point mutations in the helicase domain of the NS3 protein enhance dengue virus replicative capacity in human monocyte-derived dendritic cells and circumvent the type I interferon response.

Dengue is the most prevalent arboviral disease worldwide. The outcome of the infection is determined by the interplay of viral and host factors. In the present study, we evaluated the cellular response of human monocyte-derived DCs (mdDCs) infected with recombinant dengue virus type 1 (DV1) strains carrying a single point mutation in the NS3hel protein (L435S or L480S). Both mutated viruses infect and replicate more efficiently and produce more viral progeny in infected mdDCs compared with the parental, non-mutated virus (vBACDV1). Additionally, global gene expression analysis using cDNA microarrays revealed that the mutated DVs induce the up-regulation of the interferon (IFN) signalling and pattern recognition receptor (PRR) canonical pathways in mdDCs. Pronounced production of type I IFN were detected specifically in mdDCs infected with DV1-NS3hel-mutated virus compared with mdDCs infected with the parental virus. In addition, we showed that the type I IFN produced by mdDCs is able to reduce DV1 infection rates, suggesting that cytokine function is effective but not sufficient to mediate viral clearance of DV1-NS3hel-mutated strains. Our results demonstrate that single point mutations in subdomain 2 have important implications for adenosine triphosphatase (ATPase) activity of DV1-NS3hel. Although a direct functional connection between the increased ATPase activity and viral replication still requires further studies, these mutations speed up viral RNA replication and are sufficient to enhance viral replicative capacity in human primary cell infection and circumvent type I IFN activity. This information may have particular relevance for attenuated vaccine protocols designed for DV.

Increasing genetic variability in black oats using gamma irradiation.

The black oat (Avena strigosa Schreb) is commonly used for forage, soil cover, and green manure. Despite its importance, little improvement has been made to this species, leading to high levels of genotypic disuniformity within commercial cultivars. The objective of this study was to evaluate the efficiency of different doses of gamma rays [(60)Co] applied to black oat seeds on the increase of genetic variability of agronomic traits. We applied doses of 0, 10, 50, 100, and 200 Gy to the genotype ALPHA 94087 through exposure to [(60)Co]. Two experiments were conducted in the winter of 2008. The first aimed to test forage trait measurements such as plant height, dry matter yield, number of surviving tillers, and seedling stand. The second test assessed seed traits, such as yield and dormancy levels. Gamma irradiation seems not to increase seed yield in black oats, but it was effective in generating variability for the other traits. Tiller number and plant height are important selection traits to increase dry matter yield. Selection in advanced generations of mutant populations can increase the probability of identifying superior genotypes.

Selfing confirmation in sugarcane by using simple sequence repeat markers: an individual reciprocal recurrent selection scheme.

Superior inbred clones selected in S1 families can integrate an individual reciprocal recurrent selection program in sugarcane by eliminating the genetic load of the population and exploring superior hybrid combinations. Molecular markers can be used for reliable identification of the true selfing-derived clones in these S1 populations. The objective of this study was to confirm true self-fertilized individuals in sugarcane families using microsatellite markers aimed at the use of self-fertilized plants in an individual reciprocal recurrent selection strategy. Self-fertilized individuals from five cultivars were genotyped with eight simple sequence repeat (SSR) markers. The markers generated 62 polymorphic markers, with an average of seven polymorphic alleles across the cultivars tested. Three loci revealed highly informative bands and were used to assess the level of selfing in five S1 families. Selfing in these families ranged from 71.7 to 97.6%. The SSR loci provide a reliable and accurate method to identify S1 progenies in sugarcane crosses and can be used as a tool to assist selection strategies in sugarcane breeding programs.

Effect of compression load and temperature on thermomechanical tests for gutta-percha and Resilon®.

AIM: To analyse a method used to evaluate the thermomechanical properties of gutta-percha and Resilon(®) at different temperatures and compression loads. METHODOLOGY: Two hundred and seventy specimens measuring 10 mm in diameter and 1.5 mm in height were made from the following materials: conventional gutta-percha (GCO), thermoplastic gutta-percha (GTP) and Resilon(®) cones (RE). After 24 h, the specimens were placed in water at 50 °C, 60 °C or 70 °C for 60 s. After that, specimens were placed between two glass slabs, and loads weighing 1.0, 3.0 or 5.0 kg were applied. Images of the specimens were digitized before and after the test and analysed using imaging software to determine their initial and final areas. The thermomechanical property of each material was determined by the difference between the initial and final areas of the specimens. Data were subjected to anova and SNK tests at 5% significance. To verify a possible correlation between the results of the materials, linear regression coefficients (r) were calculated. RESULTS: Data showed higher flow area values for RE under all compression loads at 70 °C and under the 5.0 kg load at 60 °C (P < 0.05). Regarding gutta-percha, GTP showed higher flow under loads weighing 3.0 and 5.0 kg, at 60 and 70 °C (P < 0.05). GCO presented higher flow at 70 °C with a load of 5.0 kg. Regression analyses showed a poor linear correlation amongst the results of the materials under the different experimental conditions. CONCLUSION: Gutta-percha and Resilon(®) cones require different compression loads and temperatures for evaluation of their thermomechanical properties. For all materials, the greatest flow occurred at 70 °C under a load of 5.0 kg; therefore, these parameters may be adopted when evaluating endodontic filling materials.

Efficient calculation of higher-order optical waveguide dispersion.

An efficient numerical strategy to compute the higher-order dispersion parameters of optical waveguides is presented. For the first time to our knowledge, a systematic study of the errors involved in the higher-order dispersions' numerical calculation process is made, showing that the present strategy can accurately model those parameters. Such strategy combines a full-vectorial finite element modal solver and a proper finite difference differentiation algorithm. Its performance has been carefully assessed through the analysis of several key geometries. In addition, the optimization of those higher-order dispersion parameters can also be carried out by coupling to the present scheme a genetic algorithm, as shown here through the design of a photonic crystal fiber suitable for parametric amplification applications.

Is the 5-HTTLPR polymorphism associated with bipolar disorder or with suicidal behavior of bipolar disorder patients?

The serotonin transporter gene has a 44 bp insertion/deletion polymorphism within the promoter region (5-HTTLPR) with two allelic forms, the long (L) and the short (S) variants. Association between the low-activity S variant and bipolar disorder (BPD) has been shown but its replication has not been consistent. It has also been described as an association between the S allele and suicidal behavior. Since suicidal behavior is a rather frequent event in BPD, an important question is whether suicidality, instead of bipolarity itself, could be related to S allele. We assessed 351 subjects (167 bipolar inpatients and 184 healthy controls). Diagnosis was conducted by a psychiatrist using a structured interview (MINI-PLUS), according to DSM-IV criteria. Suicidal behavior was assessed using a semi-structured instrument and a review of medical records. Genotyping of the 5-HTTLPR was performed using PCR. There were 77 patients with a history of previous suicide attempts. Bipolar patients and healthy controls showed comparable genotypic and allelic frequencies. Patients carrying the S allele made violent suicide attempts more frequently (chi(2) = 20.2; P = 0.0001) and made more suicide attempts (t = 2.6; P = 0.01). We were able to show an association between the S allele and suicidal behavior but not with BPD. Our data suggest that a phenotypic stratification, taking into account the suicidal behavior history, is of pivotal importance when performing association studies between BPD and 5-HTTLPR genotypes, which could explain previous contradictory results.

Survival and prognostic factors of patients treated for Stage I to Stage III endometrial carcinoma in a reference cancer center in Southern Brazil.

PURPOSE: To describe two- and five-year survival of patients with Stage I to III endometrial carcinoma and to identify prognostic factors. STUDY DESIGN: Concurrent cohort study. PATIENTS AND METHODS: Seventy-two patients were operated on by the same surgeon and followed up for at least two years. All the histopathological examinations were performed by the same pathologist. Survival was analyzed by the Kaplan-Meier method. Age, body mass index, tumor grade, myometrial invasion, histological type and stage were correlated with death. RESULTS: Overall survival at two and five years was 90.2% and 81.4%, respectively. By bivariate analysis, FIGO stage, myometrial invasion, tumor grade, histology, adnexal and/or lymph node metastasis and age were significant predictors of death (p < 0.05). Multivariate analysis revealed significant associations with death: FIGO Stage III (p = 0.001), histological type other than endometrioid (p = 0.027) and age 70 or more (p = 0.04). CONCLUSION: Endometrial carcinoma Stage III patients, histological types other than endometrioid and age 70 years or more are at significant risk for death.

Qualea grandiflora, a Brazilian "Cerrado" medicinal plant presents an important antiulcer activity.

Qualea grandiflora is one of the species widely used in folk medicine to treat gastric ulcers in Cerrado of the central region of Brazil. The hydroalcoholic extract of bark (HE) of Qualea grandiflora was investigated for their ability to prevent and heal lesions in the gastric mucosa. The oral administration of HE exhibited antiulcer activity decreasing the ulcerative index induced by HCl/ethanol solution, indomethacin/bethanechol and stress. In the Shay model, results showed that HE (p.o.) only reduced the severity of gastric lesions without effects on pH, gastric acidity or volume. When given by intraduodenal route, HE changed the pH, but did not modify the other parameters of the gastric juice. These data were in accordance with those obtained when HE was administered orally for 14 days after gastric ulcers were induced by acetic acid in rats. HE presented healing process in subacute gastric ulcer induced by acetic acid in rats. Moreover, histological examinations showed the simple columnar epithelium, lamina propria with simple branched tubular glandules with dilated lumen and large amounts of mucus secretion. Phytochemical investigation of HE led to the detection of terpenes, steroids, saponins, phenolic compounds and tannins in this extract, which may be involved in the observed activity.

Transient and widespread astroglial activation in the brain after a striatal 6-OHDA-induced partial lesion of the nigrostriatal system.

The authors have previously described astroglial activation in the ipsilateral nigrostriatal system and ventral tegmental area following small doses of 6-hydroxydopamine (6-OHDA) injected unilaterally in the striatum. This article further evaluated astroglial reactivity in several brain regions after striatal 6-OHDA-induced punctate lesion in the nigrostriatal pathway. Adult male Wistar rats received a unilateral stereotaxical injection of the 6-OHDA (8 microg/4 microl) in the neostriatum and sacrificed 1 or 3 weeks later. Control animals received only solvent. Immunohistochemistry was employed for visualization of the tyrosine hydroxylase (TH), marker for dopamine cells, and glial fibrillary acidic protein (GFAP), marker for astrocytes. TH immunoreactive terminals disappeared in the striatum close to the injection site and a disappearance of a small number of a defined population of dopamine cell bodies was observed in the ipsilateral pars compacta of the substantia nigra (SNc). No dopamine lesion was detected in the contralateral nigrostriatal pathway. Astroglial reaction was seen close to the lesion in the neostriatum and in the ipsilateral SNc of the 1 week 6-OHDA lesioned rats. Specific stereological tools employing point intercepts and rotator, revealed an increased presence of reactive astrocytes in many forebrain regions like frontal, parietal and piriform cortex, septum, neostriatum and SNc, bilaterally, and also corpus callosum after 1 week of 6-OHDA injection. The astroglial activation was characterized by increases in the size of the cell body and/or processes. Astrocytic reaction was found only in the ipsilateral nigrostriatal pathway by 3 weeks of 6-OHDA, a slight activation also remaining in the ipsilateral septum and piriform cortex. Astrocytic reaction was seen in the solvent-injected rats only in the neostriatum close to the needle track. The transient widespread astroglial reaction observed in many brain regions following a striatal injection of 6-OHDA may represent a global paracrine trophic response in the brain.

Avaliação dietética de amidos pelas respostas glicêmica e insulinêmica em cães / Dietary evaluation of starches through glycemic and insulinemic responses in healthy dogs

Avaliaram-se, em cães saudáveis, os efeitos da fonte e do tipo de processamento do amido presente no alimento sobre as concentrações de insulina e glicose plasmáticas pós-prandiais. Foram utilizados oito cães da raça Beagle, adultos, alimentados com quatro regimes: amido cru de trigo (ACT), amido gelatinizado de trigo (AGT), amido cru de mandioca (ACM) e amido gelatinizado de mandioca (AGM). As amostras de sangue foram coletadas mediante punção da veia braquial aos 0, 70, 140, 250 e 360 minutos pós-prandiais. O regime não influenciou a glicemia, mas alterou a concentração sangüínea de insulina (P<0,05). Na área total sob a curva insulinêmica, o regime ACT não diferiu do AGM e foi superior aos regimes AGT e ACM, em razão da viscosidade do AGT e da resistência ao ataque enzimático do ACM. Os resultados sugerem que o regime AGT deve ser preferido aos demais por minimizar a resposta insulínica pós-prandial e manter a euglicemia.

Age structure and abundance in populations of muscoid flies from a poultry facility in Southeast Brazil.

Muscina stabulans, M. domestica, Chrysomya putoria, C. megacephala and Stomoxys calcitrans were the most abundant muscoid flies captured in a poultry facility in southeastern Brazil. We examined the gonadotrophic profiles of the females caught at different sites and different times and found that Mu. stabulans and M. domestica, the predominant species, presented similar gonadotrophic profiles only when captured on the manure under the cages, but very different and sometimes opposite gonadotrophic profiles when sampled from wooden posts, vegetation or electric cords. We also determined sex ratios and relative abundance for these two species and found significant differences between them. More than 50% of the females of both species of Chrysomya captured on manure carried eggs or exhibited signs of recent oviposition. The vast majority of S. calcitrans presented ovaries with eggs or signs of recent oviposition. A small proportion of them had ovaries in the recent emerged condition. Our data on ovarian stages, sex ratio and relative abundance allowed us to associate different gonadotrophic profiles with each site and characterize each site as a resting, ovipositing or mating site.

Simulium spp. control program in Rio Grande do Sul, Brazil.

Insects of the Simuliidae family have been the object of control in Rio Grande do Sul since the 70s. Their constant attacks became a social-economical problem as well as a problem of Public Health, with serious consequences to men and to the economy of the areas in which the insects develop. At first, the control was done with a chemical larvicide Themephos ABATE 500 E, but an imperfect measuring of outflow to determine the quantity of the product made Simulium spp. resistant to it. From 1983 on, following a study of a new method for the outflow measuring, we started to use a biological larvicide Bacillus thuringiensis serovar israelensis based. The biological control uses the new method in 36.4% of the state area, assisting about 3,500,000 inhabitants.