Mechanistic study of the differences in lactic acid bacteria resistance to freeze- or spray-drying and storage.

Lactobacillus delbrueckii subsp. bulgaricus and Lactiplantibacillus plantarum are two lactic acid bacteria (LAB) widely used in the food industry. The objective of this work was to assess the resistance of these bacteria to freeze- and spray-drying and study the mechanisms involved in their loss of activity. The culturability and acidifying activity were measured to determine the specific acidifying activity, while membrane integrity was studied by flow cytometry. The glass transitions temperature and the water activity of the dried bacterial suspensions were also determined. Fourier transform infrared (FTIR) micro-spectroscopy was used to study the biochemical composition of cells in an aqueous environment. All experiments were performed after freezing, drying and storage at 4, 23 and 37 °C. The results showed that Lb. bulgaricus CFL1 was sensitive to osmotic, mechanical, and thermal stresses, while Lpb. plantarum WCFS1 tolerated better the first two types of stress but was more sensitive to thermal stress. Moreover, FTIR results suggested that the sensitivity of Lb. bulgaricus CFL1 to freeze-drying could be attributed to membrane and cell wall degradation, whereas changes in nucleic acids and proteins would be responsible of heat inactivation of both strains associated with spray-drying. According to the activation energy values (47-85 kJ/mol), the functionality loss during storage is a chemically limited reaction. Still, the physical properties of the glassy matrix played a fundamental role in the rates of loss of activity and showed that a glass transition temperature 40 °C above the storage temperature is needed to reach good preservation during storage. KEY POINTS: • Specific FTIR bands are proposed as markers of osmotic, mechanic and thermal stress • Lb. bulgaricus CFL1 was sensitive to all three stresses, Lpb. plantarum WCFS1 to thermal stress only • Activation energy revealed chemically limited reactions ruled the activity loss in storage.

Disparities in HIV continuum of care in the paediatric population: A real-life study in Brazil.

INTRODUCTION: Paediatric HIV follow-up is challenging, and treatment indicators are markedly far from Joint United Nations Programme on HIV/AIDS (UNAIDS) goals. In this study, we describe the 2019 Brazilian HIV cascade according to age categories and sociodemographic variables and address temporal trends between 2009 and 2019. METHODS: We obtained data from the Brazilian Ministry of Health monitoring database. Cascade outcomes included retention in care, antiretroviral use, and viral suppression. We assessed the effect of age on timely initiation of antiretroviral treatment (ART; initiation with CD4+ T-cell count ≥350 cells/mm3 or a first ART dispensation ≤30 days after the first CD4+ T-cell measurement) and detectable HIV viral load (>50 copies/mL) in univariable and multivariable analysis adjusted for sex, race, and social vulnerability index (SVI). Temporal trends in timely ART initiation and viral suppression were evaluated graphically. RESULTS: Among 771 774 people living with HIV, those in the youngest age categories had poorer indicators in the care cascade. Those in younger age groups, those with higher SVI, and those declaring Black and native Brazilian race/ethnicity had higher odds of having detectable viral load and delayed ART initiation. Although children living with HIV tend to start ART with higher CD4+ T-cell counts, time-series analysis suggests that improvements in treatment indicators seen in the adult population are not observed in the paediatric population. CONCLUSION: Our results highlight the challenges faced by children and adolescents living with HIV in achieving UNAIDS goals. Lower access to ART among children is a central barrier to improved paediatric care.

Relationship between the colours of the rivers in the Amazon and the incidence of malaria.

BACKGROUND: Malaria is transmitted by different Anopheles species. In Brazil, the disease is concentrated in the Amazon region. Rivers play an important role in the life cycle of malaria since the vector reproduces in aquatic environments. The waters of the rivers in the Amazon have distinct chemical characteristics, which affect the colour of the water and therefore, the study analysed whether the colour of the waters of the rivers have an on influence the distribution of malaria. The goal of the study was to correlate the different colourations of the water (black, white and mixed water) and the malaria incidence in 50 municipalities of the Amazonas state, Brazil, and then test hypotheses about the characteristics of the colour of the rivers and disease incidence. METHODS: This study was conducted for a period of seventeen years (2003-2019) in 50 municipalities in the state of Amazonas, Brazil. A conditionally Gaussian dynamic linear model was developed to analyse the association of malaria incidence and three types of river colour: white, black and mixed. RESULTS: The analyses indicate that the distribution of malaria is related to the colouration of the rivers. The results showed that places located near black-water rivers have a higher malaria incidence when compared to places on the banks of white-water rivers. CONCLUSIONS: Historically, the hydrological regime has played an important role in the dynamics of malaria in the Amazon, but little is known about the relationship between river colours and the incidence of the disease. This research was carried out in a region with hydrographic characteristics that were heterogeneous enough to allow an analysis that contrasted different colours of the rivers and covered almost the whole of the state of Amazonas. The results help to identify the places with the highest risk of malaria transmission and it is believed that they will be able to contribute to more precise planning of actions aimed at controlling the disease in the region.

Multiobjective optimization of frozen and freeze-dried Lactobacillus delbrueckii subsp. bulgaricus CFL1 production via the modification of fermentation conditions.

AIM: This study investigates the individual and combined effects of fermentation parameters for improving cell biomass productivity and the resistance to freezing, freeze-drying, and freeze-dried storage of Lactobacillus delbrueckii subsp. bulgaricus CFL1. METHODS AND RESULTS: Cells were cultivated at different temperatures (42°C and 37°C) and pH values (5.8 and 4.8) and harvested at various growth phases (mid-exponential, deceleration, and stationary growth phases). Specific acidifying activity was determined after fermentation, freezing, freeze-drying, and freeze-dried storage. Multiple regression analyses were performed to identify the effects of fermentation parameters on the specific acidifying activity losses and to generate the corresponding 3D response surfaces. A multiobjective decision approach was applied to optimize biomass productivity and specific acidifying activity. The temperature positively influenced biomass productivity, whereas low pH during growth reduced the loss of specific acidifying activity after freezing and freeze-drying. Furthermore, freeze-drying resistance was favored by increased harvest time. CONCLUSIONS: Productivity, and freezing and freeze-drying resistances of L. delbrueckii subsp. bulgaricus CFL1 were differentially affected by the fermentation parameters studied. There was no single fermentation condition that improved both productivity and resistance to freezing and freeze-drying. Thus, Pareto fronts were helpful to optimize productivity and resistance, when cells were grown at 42°C, pH 4.8, and harvested at the deceleration phase.

Insights into the mechanisms of L. salivarius CECT5713 resistance to freeze-dried storage.

Ligilactobacillus salivarius is a lactic acid bacterium exhibiting several health benefits. However, it is sensitive to freeze-drying and storage in the dried state, thus limiting its commercial exploitation. Our objective was to identify markers of cell resistance by applying multiscale characterization to L. salivarius CECT5713 cell populations exhibiting different resistance to freeze-dried storage. Cells were produced under two different sets of production conditions differing in the culture parameters (temperature, neutralizing solution, and harvesting time) and the protective formulation composition. The culturability, membrane integrity, and cell biochemical composition assessed by Fourier transform infrared (FTIR) micro-spectroscopy were evaluated after freezing, freeze-drying, and subsequent storage at 37 °C. Membrane properties (fatty acid composition, membrane fluidity, and phospholipid organization), as well as matrix physical properties (glass transition temperature and water activity), were determined. The most resistant cells to freeze-dried storage exhibited the highest cyclic fatty acid content and the most rigid membrane. Freeze-drying and storage induced damage to membrane integrity, proteins, nucleic acids, and constituents of the peptidoglycan cell wall. From the FTIR spectra analysis, we propose the minimization of the variations of the 1058 and 1714 cm-1 vibration bands (that arise mainly from symmetric C-O-C stretching and CO stretching, respectively) induced by the freeze-drying process as a marker of storage stability. We confirmed that a matrix with a glass transition temperature at least 50 °C higher than the storage temperature is crucial for L. salivarius CECT5713 storage stability. In addition, this work explored promising FTIR methods for a better understanding of the protection mechanisms involved.

Recurrence in Cervical High-Grade Squamous Intraepithelial Lesion: The Role of the Excised Endocervical Canal Length-Analysis of 2,427 Patients.

OBJECTIVE: This study aimed to evaluate the influence of the excised canal length on relapse rates of cervical high-grade squamous intraepithelial lesion (HSIL) treated by loop electrosurgical excision procedure and to find a cut-off point, above which lower recurrence rates could be observed, with low probability of compromising future obstetric outcome, and the relationship with other individual factors related to HSIL recurrence. METHOD: This was a retrospective cohort study of 2,427 women diagnosed with cervical intraepithelial neoplasia CIN2+ who underwent cervical conization using the high-frequency loop electrosurgical excision procedure surgery technique, to analyze the role of endocervical canal length associated with individual factors in the recurrent disease after CIN2+ treatment and determine a cut-off point for the excised canal length needed to decrease the risk of disease relapse. RESULTS: In 2,427 cases, the relapse rate of HSIL treated was 12%. Compromised margins of conization, HIV+, and endocervical canal length were related directly to relapses ( p < .001). The cut-off point, by receiver operating characteristic curve, to calculate the endocervical canal length related to relapses was 1.25 cm of canal excised. Canal length of less than 1.25 cm increased the recurrence rate 2.5 times. Compromised margins and HIV+ increased recurrence rates by more than 5 times. CONCLUSION: Cervical HSIL recurrence was directly related to the endocervical canal length: excised canal length of 1.25 cm or more decreases recurrence rate; HIV and compromised margins increase the chance of recurrence by more than 5 times.

Association of physical performance and sarcopenia with use of health services in elderly people living in rural riverside areas in the Amazon: a cross-sectional study.

INTRODUCTION: Access is considered one of the necessary conditions for achieving effectiveness and quality in health services. However, it represents a complex construct, with several interpretations, and can be understood as the ease or degree of difficulty with which people obtain effective and timely care. Barriers to access can be related to individual characteristics and those of health systems and services. Regarding elderly people living in rural riverine localities, these limitations are exacerbated due to the territorial dispersion of households and the difficulty of obtaining the necessary care near their homes. The aim of this study was to describe and test the association of sarcopenia and physical performance with primary healthcare attributes and the use of health services by elderly people living in rural riverside areas in the Amazon, Brazil. METHODS: This cross-sectional observational study was carried out in households with individuals aged 60 years or older living in nine communities located on the left bank of the Negro River, in the rural riverside area of the municipality of Manaus, Amazonas, Brazil. The study evaluated socioeconomic and demographic conditions, health services utilization and the primary care attributes related to the use of and access to services, assessed by components of the Primary Care Assessment Tool instrument (PCATool-Brazil), a reduced version validated for Brazilian adult users. Physical performance was assessed using the Short Physical Performance Battery scale, and handgrip strength was also assessed, according to a dynamometer. The Sarcopenia Formulary (SARC-F) and calf circumference (CC) were used to assess sarcopenia (SARC-CalF). The association of sarcopenia and physical performance with the study outcomes was evaluated using hierarchical logistic regression for health services utilization (having had a medical consultation in the last year), and hierarchical linear regression for the continuous outcomes of the PCATool-Brazil (total score and each of the domains). The sociodemographic variables were inserted in model 1 and the clinical variables in model 2. Variables with p<0.20 were kept in the models. RESULTS: A total of 98 elderly people (55.1% men; mean age 70±7.4 years) were included in the study. Low physical performance and suggestive signs of sarcopenia were observed in 52.5% and 28.9% of the study participants, respectively. Elderly with better physical performance reported more health services utilization (odds ratio (OR)=1.37; 95% confidence interval (CI)=1.03-1.81) and higher scores in the affiliation (β=1.67; 95%CI=0.37-2.98), utilization (β=1.19; 95%CI=0.06-2.33) and longitudinality (β=0.99; 95%CI=0.09-1.90) domains of the PCATool-Brazil. CONCLUSION: The study findings showed high prevalence of impairment in physical performance and suggestive signs of sarcopenia in elderly people living in the studied rural riverside localities. Better physical performance was associated with use of health services in the previous year and with better evaluation of some primary care attributes.

MASTREE+: Time-series of plant reproductive effort from six continents.

Significant gaps remain in understanding the response of plant reproduction to environmental change. This is partly because measuring reproduction in long-lived plants requires direct observation over many years and such datasets have rarely been made publicly available. Here we introduce MASTREE+, a data set that collates reproductive time-series data from across the globe and makes these data freely available to the community. MASTREE+ includes 73,828 georeferenced observations of annual reproduction (e.g. seed and fruit counts) in perennial plant populations worldwide. These observations consist of 5971 population-level time-series from 974 species in 66 countries. The mean and median time-series length is 12.4 and 10 years respectively, and the data set includes 1122 series that extend over at least two decades (≥20 years of observations). For a subset of well-studied species, MASTREE+ includes extensive replication of time-series across geographical and climatic gradients. Here we describe the open-access data set, available as a.csv file, and we introduce an associated web-based app for data exploration. MASTREE+ will provide the basis for improved understanding of the response of long-lived plant reproduction to environmental change. Additionally, MASTREE+ will enable investigation of the ecology and evolution of reproductive strategies in perennial plants, and the role of plant reproduction as a driver of ecosystem dynamics.

Aún existen importantes vacíos en la comprensión de la respuesta reproductiva de las plantas al cambio medioambiental, en parte, porque su monitoreo en especies de plantas longevas requiere una observación directa durante muchos años, y estos conjuntos de datos rara vez han estado disponibles. Aquí presentamos a MASTREE +, una base de datos que recopila series de tiempo de la reproducción de las plantas de todo el planeta, poniendo a disposición estos datos de libre acceso para la comunidad científica. MASTREE + incluye 73.828 puntos de observación de la reproducción anual georreferenciados (ej. conteos de semillas y frutos) en poblaciones de plantas perennes en todo el mundo. Estas observaciones consisten en 5971 series temporales a nivel de población provenientes de 974 especies en 66 países. La mediana de la duración de las series de tiempo es de 10 años (media = 12.4 años) y el conjunto de datos incluye 1.122 series de al menos dos décadas (≥20 años de observaciones). Para un subconjunto de especies bien estudiadas, MASTREE +incluye un amplio conjunto de series temporales replicadas en gradientes geográficos y climáticos. Describimos el conjunto de datos de acceso abierto disponible como un archivo.csv y presentamos una aplicación web asociada para la exploración de datos. MASTREE+ proporcionará la base para mejorar la comprensión sobre la respuesta reproductiva de plantas longevas al cambio medioambiental. Además, MASTREE+ facilitará los avances en la investigación de la ecología y la evolución de las estrategias reproductivas en plantas perennes y el papel de la reproducción vegetal como determinante de la dinámica de ecosistemas.

Insights into lactic acid bacteria cryoresistance using FTIR microspectroscopy.

Freezing is widely used for bacterial cell preservation. However, resistance to freezing can greatly vary depending on bacterial species or growth conditions. Our study aims at identifying cellular markers of cryoresistance based on the comparison of three lactic acid bacteria (LAB) exhibiting different tolerance to freezing: Carnobacterium maltaromaticum CNCM I-3298, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, and Lactobacillus delbrueckii subsp. bulgaricus CFL1. A thorough characterization of their cytoplasmic membrane properties was carried out by measuring their fatty acid composition, membrane fluidity, and lipid phase transition upon cooling from 50 to -50 °C. Vitrification temperatures of the intra- and extra-cellular compartments were also quantified by differential scanning calorimetry. Additionally, the cell biochemical characterization was carried out using a recently developed Fourier transform infrared (FTIR) micro-spectroscopic approach allowing the analysis of live bacteria in an aqueous environment. The multivariate analysis of the FTIR spectra of fresh and thawed cells enabled the discrimination of the three bacteria according to their lipid, protein, and cell wall peptidoglycan components. It also revealed freezing-induced modifications of these three cellular components and an increase in bacteria heterogeneity for the two strains of L. bulgaricus, the freeze-sensitive bacteria. No cellular damage was observed for C. maltaromaticum, the freeze-resistant bacteria. Comparison of the results obtained from the different analytical methods confirmed previously reported cryoresistance markers and suggested new ones, such as changes in the absorbance of specific infrared spectral bands. FTIR microspectroscopy could be used as a rapid and non-invasive technique to evaluate the freeze-sensitivity of LAB.

Effect of protective agents on the storage stability of freeze-dried Ligilactobacillus salivarius CECT5713.

Ligilactobacillus salivarius is a lactic acid bacterium exhibiting several health benefits but remains commercially underexploited due to its inability to survive during long-term storage in the dried state. Our objective was to study the effect of various protective molecules (maltodextrin, trehalose, antioxidants, and fructooligosaccharides), being efficient on other bacteria, on the freeze-dried stability of L. salivarius CECT5713. The culturability was evaluated after freezing, freeze-drying, and subsequent storage at 37 °C, as well as the biochemical composition of cells in an aqueous environment using Fourier transform infrared (FTIR) micro-spectroscopy. The assignment of principal absorption bands to cellular components was performed using data from the literature on bacteria. The membrane fatty acid composition was determined after freeze-drying and storage. Glass transition temperature of the liquid and freeze-dried bacterial suspensions and water activity of the freeze-dried samples were measured. The best storage stability was observed for the formulations involving maltodextrin and antioxidants. The analysis of the FTIR spectra of freeze-thawed cells and rehydrated cells after freeze-drying and storage revealed that freeze-drying induced damage to proteins, peptidoglycans of the cell wall and nucleic acids. Storage stability appeared to be dependent on the ability of the protective molecules to limit damage during freeze-drying. The inactivation rates of bacteria during storage were analyzed as a function of the temperature difference between the product temperature during sublimation or during storage and the glass transition temperature, allowing a better insight into the stabilization mechanisms of freeze-dried bacteria. Maintaining during the process a product temperature well below the glass transition temperature, especially during storage, appeared essential for L. salivarius CECT5713 storage stability. KEY POINTS: • L. salivarius CECT5713 highly resisted freezing but was sensitive to freeze-drying and storage. • Freeze-drying and storage mainly altered cell proteins, peptidoglycans, and nucleic acids. • A glassy matrix containing maltodextrin and an antioxidant ensured the highest storage stability.

Self-reported chronic back pain in the elderly living in rural riverine areas in the Amazon.

INTRODUCTION: The present study aimed to estimate the prevalence of self-reported chronic low back pain and to identify the associated factors in elderly people living in rural riverside areas in the Amazon. METHODS: Data from a cross-sectional home-based survey performed in 38 riverside rural locations along the left bank of the Negro River, in the municipality of Manaus, Amazonas, Brazil, were analyzed. The selected elderly people answered a questionnaire comprising items related to the living conditions, socioeconomic and demographic characteristics, health status and utilization of health services. The outcome was self-reported chronic back pain or problem. The association between the outcome and the independent variables was assessed using logistic regression, estimating the odds ratios and respective 95% confidence intervals (CIs). RESULTS: One hundred and six residents aged 60 years or older out of a total of 277 were included in the study. The prevalence of chronic back pain among the elderly residents was 57.5% (63.3% among men and 50.0% among women). Most of them did not have any treatment related to their chronic condition (77.0%). Occupation in activities other than agricultural activities was protective for the outcome (odds ratio (OR)=0.1, 95%CI=0.0-0.9). The chronic low back pain was also associated with worse self-perceived health (OR=10.3, 95%CI=1.6-67.5). CONCLUSION: The high prevalence of chronic low back pain in elderly people living in rural riverside areas, along with limited access to treatment and the different impacts on the performance of daily activities and the quality of life, reveals the relevance of this condition as a public health problem.

Monoclonal Antibodies against Cell Wall Chitooligomers as Accessory Tools for the Control of Cryptococcosis.

Therapeutic strategies against systemic mycoses can involve antifungal resistance and significant toxicity. Thus, novel therapeutic approaches to fight fungal infections are urgent. Monoclonal antibodies (MAbs) are promising tools to fight systemic mycoses. In this study, MAbs of the IgM isotype were developed against chitin oligomers. Chitooligomers derive from chitin, an essential component of the fungal cell wall and a promising therapeutic target, as it is not synthesized by humans or animals. Surface plasmon resonance (SPR) assays and cell-binding tests showed that the MAbs recognizing chitooligomers have high affinity and specificity for the chitin derivatives. In vitro tests showed that the chitooligomer MAbs increased the fungicidal capacity of amphotericin B against Cryptococcus neoformans. The chitooligomer-binding MAbs interfered with two essential properties related to cryptococcal pathogenesis: biofilm formation and melanin production. In a murine model of C. neoformans infection, the combined administration of the chitooligomer-binding MAb and subinhibitory doses of amphotericin B promoted disease control. The data obtained in this study support the hypothesis that chitooligomer antibodies have great potential as accessory tools in the control of cryptococcosis.

Presence of trypanosomatids, with emphasis on Leishmania, in Rodentia and Didelphimorphia mammals of a rural settlement in the central Amazon region.

BACKGROUND: Trypanosomatids are widespread and cause diseases - such as trypanosomiasis, sleeping sickness, Chagas disease, and cutaneous and visceral leishmaniasis - in animals and humans. These diseases occur in both rural and urban regions due to unplanned growth and deforestation. Thus, wild and synanthropic reservoir hosts living in residential areas are risk factors. OBJECTIVE: We aimed to evaluate the diversity of small mammals (rodents and marsupials), and the occurrence of trypanosomatids, especially Leishmania, in the rural settlement of Presidente Figueiredo, Amazonas. METHODS: Animals were collected using Sherman, Tomahawk, and Pitfall traps along 16 trails in four landscapes: continuous forest, forest with planting, planting, and peridomiciliar. Leishmania sp. was detected in liver samples by polymerase chain reaction targeting kDNA. FINDINGS: Diversity was higher in forests with planting and lower around residences. In total, 135 mammals (81 rodents and 54 marsupials covering 14 genera) were captured. Rodents presented infection rates (IR) of 74% and marsupials of 48%. Rodents in domicile landscapes presented a higher IR (92.9%), while marsupials showed a higher IR in forests (53.3%). MAIN CONCLUSIONS: The results suggest high prevalence of trypanosomatids across 12 mammalian genera possibly involved as reservoir hosts in the enzootic transmission of leishmaniasis in the Amazon's rural, peridomiciliar landscape.

Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans.

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

Macrophage profile and homing into breast milk in response to ongoing respiratory infections in the nursing infant.

Studies have shown that immune components of human milk can be changed during an infection in the nursing infant. Macrophages are abundant in human milk and they are classified into inflammatory (CD16-) and noninflammatory (CD16+) subsets. This study investigated CD16+ and CD16- macrophage homing into breast milk in response to ongoing infections in nursing infants. Peripheral blood and mature milk were collected from 33 healthy mothers of nursing infants with respiratory infections (Group I) and from 26 healthy mothers of healthy nursing infants (Group H). Blood and milk total, CD16- and CD16+ monocyte (Mo)/macrophage (Mφ) subsets, respectively, and CCR2 and CX3CR1 expression and cytokine levels were analyzed by flow cytometry. CCL2 and CX3CL1 were quantified by ELISA and cytokines by flow cytometry in serum and milk. There was an increase of total and CD16+ Mφ, and, also a decrease of CD16- Mφ frequencies in maternal milk from Group I compared to Group H, but absolute numbers analyses showed higher numbers of all subpopulations of milk Mφ in Group I compared to Group H. Higher numbers of CX3CR1+CD16+ and double-staining of CCR2 and CX3CR1 in both CD16+ and CD16- cells were observed in milk during infant infection, which weren't observed in the blood. CCR2 expression was hardly found in milk CD16- Mφ in both groups. CCL2 and CX3CL1 were both higher in milk than in blood from both groups, but Group I showed higher levels of these chemokines in milk than Group H. Breast milk showed higher IL-6 and IL-8 concentrations than serum, and infant infection caused an increase in these cytokines only in milk. Our findings suggest that milk Mφ profiles are different from blood Mo, and the ongoing infection in the nursing infant could change milk Mφ to a more anti-inflammatory profile compared to that in the healthy group, possibly as an additional strategy of infant protection.

The Overlooked Glycan Components of the Cryptococcus Capsule.

Pathogenic species of Cryptococcus kill approximately 200,000 people each year. The most important virulence mechanism of C. neoformans and C. gattii, the causative agents of human and animal cryptococcosis, is the ability to form a polysaccharide capsule. Acapsular mutants of C. neoformans are avirulent in mice models of infection, and extracellularly released capsular polysaccharides are deleterious to the immune system. The principal capsular component in the Cryptococcus genus is a complex mannan substituted with xylosyl and glucuronyl units, namely glucuronoxylomannan (GXM). The second most abundant component of the cryptococcal capsule is a galactan with multiple glucuronyl, xylosyl, and mannosyl substitutions, namely glucuronoxylomannogalactan (GXMGal). The literature about the structure and functions of these two polysaccharides is rich, and a number of comprehensive reviews on this topic are available. Here, we focus our discussion on the less explored glycan components associated with the cryptococcal capsule, including mannoproteins and chitin-derived molecules. These glycans were selected for discussion on the basis that i) they have been consistently detected not only in the cell wall but also within the cryptococcal capsular network and ii) they have functions that impact immunological and/or pathogenic mechanisms in the Cryptococcus genus. The reported functions of these molecules strongly indicate that the biological roles of the cryptococcal capsule go far beyond the well-known properties of GXM and GXMGal.

FTIR micro-spectroscopy using synchrotron-based and thermal source-based radiation for probing live bacteria.

Fourier transform infrared (FTIR) spectroscopy has proven to be a non-invasive tool to analyse cells without the hurdle of employing exogenous dyes or probes. Nevertheless, the study of single live bacteria in their aqueous environment has long remained a big challenge, due to the strong infrared absorption of water and the small size of bacteria compared to the micron-range infrared wavelengths of the probing photons. To record infrared spectra of bacteria in an aqueous environment, at different spatial resolutions, two setups were developed. A custom-built attenuated total reflection inverted microscope was coupled to a synchrotron-based FTIR spectrometer, using a germanium hemisphere. With such a setup, a projected spot size of 1 × 1 µm2 was achieved, which allowed spectral acquisition at the single-cell level in the 1800-1300 cm-1 region. The second setup used a demountable liquid micro-chamber with a thermal source-powered FTIR microscope, in transmission geometry, for probing clusters of a few thousands of live cells in the mid-IR region (4000-975 cm-1). Both setups were applied for studying two strains of a model lactic acid bacterium exhibiting different cryo-resistances. The two approaches allowed the discrimination of both strains and revealed population heterogeneity among bacteria at different spatial resolutions. The multivariate analysis of spectra indicated that the cryo-sensitive cells presented the highest cell heterogeneity and the highest content of proteins with the α-helix structure. Furthermore, the results from clusters of bacterial cells evidenced phosphate and peptidoglycan vibrational bands associated with the cell envelope, as potential markers of resistance to environmental conditions. Graphical Abstract.

Pharmacological inhibition of pigmentation in Cryptococcus.

Melanin formation is a promising target for antifungal development. We screened a collection of 727 compounds that were previously approved for clinical use in humans for inhibition of pigmentation in Cryptococcus gattii, a lethal fungal pathogen that causes damage to both immunocompetent and immunocompromised hosts. The pyrimidine analogues flucytosine (5-fluorocytosine [5-FC]), 5-fluorouracil (5-FU) and carmofur were identified as efficient inhibitors of pigmentation in the C. gattii model. Since melanin synthesis is enzymatically catalyzed by laccase in Cryptococcus, we investigated whether inhibition of pigmentation by the pyrimidine analogues was laccase-mediated. Enzyme activity and expression of LAC genes were not involved in the effects of the pyrimidine analogues, suggesting alternative cellular targets for inhibition of pigmentation. To address this hypothesis, we screened a collection of approximately 8000 mutants of C. gattii that were produced by insertional mutation after incubation with Agrobacterium tumefaciens and identified a gene product required for the anti-pigmentation activity of 5-FC as a beta-DNA polymerase. Reduced expression of this gene affected capsule formation and urease activity, suggesting essential roles in the cryptococcal physiology. These results demonstrate a previously unknown antifungal activity of 5-FC and reveal a promising target for the development of novel antifungals.

Factors influencing the membrane fluidity and the impact on production of lactic acid bacteria starters.

Production of lactic acid bacteria starters for manufacturing food, probiotic, and chemical products requires the application of successive steps: fermentation, concentration, stabilization, and storage. Despite process optimization, losses of bacterial viability and functional activities are observed after stabilization and storage steps due to cell exposure to environmental stresses (thermal, osmotic, mechanical, and oxidative). Bacterial membrane is the primary target for injury and its damage is highly dependent on its physical properties and lipid organization. Membrane fluidity is a key property for maintaining cell functionality, and depends on lipid composition and cell environment. Extensive evidence has been reported on changes in membrane fatty acyl chains when modifying fermentation conditions. However, a deep characterization of membrane physical properties and their evolution following production processes is scarcely reported. Therefore, the aims of this mini-review are (i) to define the membrane fluidity and the methods used to assess it and (ii) to summarize the effect of environmental conditions on membrane fluidity and the resulting impact on the resistance of lactic acid bacteria to the stabilization processes. This will make it possible to highlight existing gaps of knowledge and opens up novel approaches for future investigations.

Scientific and technological contributions of Latin America and Caribbean countries to the Zika virus outbreak.

BACKGROUND: The recent Zika virus (ZIKAV) epidemics disclosed a major public health threat and a scientific and technological (S&T) challenge. The lessons learned from the S&T response of Latin America and the Caribbean (LAC) countries are critical to inform further research and guide scientific investments. The present study aimed to assess how new S&T knowledge produced and disseminated regionally can contribute to address global health challenges. METHODS: Scientometric and social network analysis methods were used to assess the LAC scientific contribution and potential technological development on ZIKAV up to December 2017. ZIKAV-related publications were retrieved from the Web of Science, Scopus, and PubMed databases. Regionally published articles were obtained from SciELO (Scientific Electronic Library Online) and LILACS (Literature in the Health Sciences in Latin America and the Caribbean) databases. Patent registries were retrieved using Orbit Intelligence and Derwent Innovation. Records from each database were individually downloaded, integrated, standardized and analyzed. RESULTS: We retrieved 5421 ZIKAV-related publications, revealing a sharp increase from 2015 onwards. LAC countries accounted for 20% of all publications and Brazil was among the top three most central countries in the global network for ZIKAV research. A total of 274 patent families backed up by experimental evidence were retrieved. Only 5% were filed by LAC assignees, all of them based in Brazil. The largest contribution of LAC research was on the clinical manifestations of the ZIKAV infection, along with vector control, which was also the main focus of patents. CONCLUSIONS: Our analysis offered a comprehensive overview of ZIKAV's research and development and showed that (i) LAC countries had a key role in generating and disseminating scientific knowledge on ZIKAV; (ii) LAC countries have expressively contributed to research on ZIKAV clinical manifestations; (iii) the Brazilian scientific community was potentially very effective in knowledge sharing and diffusion in the ZIKAV research network; (iv) Brazil was the single LAC country filing patents, mostly represented by independent inventors and low-tech patents. The paper advocates the need for a continued interdisciplinary approach to improve LAC countries ability to prevent, prepare for and control future outbreaks.