Biology subjects

Freitas, F.

Publications and source records attributed to Freitas, F..

13 recordsLinked to original sources

bioRxiv2025

Tosylation-mediated uronic acid knockout from cryoprotective FucoPol revealed the importance of polyanionicity in ice growth disruption

Negative formal charge is a critical structural trait for the expression of cryoprotection due to its strong ice growth disruption at the growing ice facet. Polyanionic polysaccharides containing uronic acids (UA) in their structure are therefore often associated with psychrophilic-like traits, such as defense against cryoinjury. However, their practical biological post-thaw benefit is convoluted amongst other contributing factors, including hydrophilic-hydrophobic balance, molecular weight, rheological properties, and structural conformation. Here, we leveraged ubiquitous (31.7% m.d.w.) and COOH-targeted (63.4% m.d.w.) leaving-group SN2 tosylation procedures, coupled with clean purification methodologies (high-MW membrane dialysis), to synthesize UA-free variants (FP-OTs18, FP-OTs96) of the cryoprotective, UA-containing polysaccharide FucoPol (FP). Tosylation was confirmed by ATR-FTIR (1602.3 cm-1 shift to 1627.9 cm-1) and 1H-NMR (brs,{delta} 7.92 ppm; brs, 7.53 ppm). Tosylated derivatives exhibited reduced solution conductivity ({Omega}=300 {micro}S/cm vs. ~800 {micro}S/cm), unchanged molecular weight (Mw), and retained biocompatibility. However, the substitution of hydrophilic, low-steric hindrance carboxyl groups with hydrophobic, high steric hindrance tosyl groups promoted a 30-40% reduction in zero-shear viscosity (0) and precluded the formation of a hypothermic gel-state due to loss of inter-chain non-covalent interactions, leading to a drastic loss of cryoprotective activity (10-70% of the original post-thaw viability observed for native FucoPol). The cryobiologically antagonistic effect of FP-OTs18 and FP-OTs96 reveals that UAs are a key player in polysaccharide-based cryopreservation, not only due to standalone beneficial polyanionicity, but also because their presence largely contributes to the expression of agonistic structural traits that elicit a cryoprotective response.

biochemistry↗

bioRxiv2025

Elucidation of the polysaccharide cryoprotection mechanism: kinetic inhibition, thermodynamic Gibbs-Thomson modulation and volumetric confinement

The emergence of gel-forming, ice-binding polysaccharides as potential candidates in cryobiology and the discovery of new structure-function relationships has fueled a knowledge convergence effort. Several polysaccharides have shown strong biological post-thaw benefits in cell cryopreservation despite some expressing contradictory ice growth anticipation, the main source of cryoinjury. The bio-based fucose-rich polysaccharide FucoPol, a current model under our scope of expertise, has further demonstrated crystal size reduction, thermal hysteresis, nucleation anticipation, nucleation stochastic narrowing and Gibbs-Thomson growth modulation effects, the latter similar to a type I antifreeze protein, in different thermodynamic settings: bulk vs. directional freezing; isobaric vs. isochoric systems; and sterile vs. biological media. Here, we undergo a critical reiteration of the consortium of findings over the years on cryoprotective polysaccharide research, rationalize several heuristic models to explain a dual nucleation behavior scenario and put forth a unifying theory to explain in which subset of conditions optimal cryoprotection may emerge from bio-based polysaccharides. We argue that gel-forming, ice-binding polysaccharides that show cryoprotective traits by anticipating nucleation and ice growth act by a combination of kinetic hampering of molecular diffusion (concentration effect); Gibbs-Thomson specific ice binding (templating effect) that induces growth modulation and size reduction; and indulge in the formation of a gel architecture of defined porosity (mesh size effect), the main initiator of a predominant pro-nucleation setting, increased stochastic determinism and the annihilation of large r* nuclei that elicits a small-nuclei survivorship bias. Classical Nucleation Theory formalisms support this hypothesis under the circumstance that a change in system state, initiated by a sol-gel transition near hypothermia, must exist to drive a meaningful shift in system energetics that explains the ANTI/PRO nucleation duality observed. The molecular fractioning of kinetically hindered bulk water into fractionally partitioned gel pores reduces system scale by a mesh size constant and enables the Gibbs-Thomson ice-binding affinity condition to be satisfied when r = rGT. A decreased nucleation energy barrier and maximal r* constraint thus invokes a predominant pro-nucleating system by enhancing nucleation susceptibility.

biophysics↗

bioRxiv2025

Polysaccharides in cryopreservation: multidimensional systematic review of extremophilic traits and the role of selective pressure in structure-function relationships

Cryopreservation of biological matter has accumulated apex biomedical interest for its potential in elongating the shelf-life of biological matter in a state of suspended animation. In Nature, extremophilic microorganisms have an outstanding ability of surviving in habitats where extreme cold, heat, salinity and acidity defy the established boundaries for life. Through Darwinian selective adaptation, they have developed biochemical defense strategies to counteract lethal stimuli. Here, we have compiled an extremophilic EPS structure-function relationship database (XPOL-DB) which aggregates reports on 145 extremophilic and mesophilic EPS, for a total of 128 biochemical and establishes the psychrophilic chemical profile for cold adaptation. Psychrophilic EPS are highly-branched, polyanionic, elongated structures of increased flexibility and molecular weight (16-300 MDa), with predominant expression of polar monomers (GalNAc, GalA, GlcNAc, GlcA) - compared to the linear, rigid, neutral thermophilic EPS. This critical analysis revealed the significant EPS similarity between psychrophiles and halophiles suggests ice growth and extreme salinity are rooted in a shared mechanism of physical membrane destabilization, for which similar chemical traits can dualistically imbue freeze and salt tolerance. Psychrophiles are exciting testbeds for the mapping of how extreme cold selects for cryobiological EPS adaptation; and can fuel reverse engineering efforts to design optimal bio-based, non-cytotoxic cryoprotectant polysaccharides.

molecular biology↗

bioRxiv2025

Relaxation of cardiac pericytes by GLP-1 activating KATP channels mediates remote ischaemic preconditioning cardioprotection

Failure to reperfuse the coronary microvasculature ("no-reflow") affects up to 50% of patients after unblocking a coronary artery that caused ischaemia and acute myocardial infarction. No-reflow is associated with reduced left ventricular ejection fraction, increased infarct size and death. We have established that no-reflow results from cardiac pericytes constricting coronary capillaries, and that pharmacologically relaxing pericytes reduces no-reflow. Remote ischaemic preconditioning, by briefly making a limb ischaemic, protects against cardiac ischaemic injury, and we have shown this is mediated by release of the gut hormone glucagon-like peptide 1 (GLP-1). We now demonstrate that, by releasing GLP-1, remote ischaemic preconditioning reduces pericyte-mediated coronary capillary constriction and no-reflow, and that the dilating effect of GLP-1 on coronary capillaries is abolished by block or genetic deletion of pericyte KATP channels. These results define a brain-gut-heart pathway mediating remote ischaemic cardioprotection, and suggest pharmacological therapies to reduce ischaemia-induced coronary no-reflow and improve post-infarct recovery.

physiology↗

bioRxiv2024

Expression pattern of the ST heat-stable enterotoxin gene cassettes in Vibrios superintegrons

BACKGROUNDVibrio mimicus has been associated with a cholera-like diarrhea and other gastroenteritis due to the presence of virulence factors such as the heat-stable ST enterotoxin. In spite of its relevant role in Vibrio pathogenesis, there is still a lack of information concerning ST expression dynamics and associated genomic context. OBJECTIVEthis study aimed to determine the prevalence, genomic context, and expression of the st gene found in V. cholerae and V. mimicus strains. METHODSgenome sequencing, in silico analyses of st promoter region and genomic context, and relative quantification pf st expression by real time RT-PCR were applied. FINDINGSThe st gene was found within the SI of both Vibrio species, presenting variations in their amino acid sequences. The in silico analysis of the st 5UTR identified different promoter versions among them. Significant differences were observed in the st expression level, which varied from 10X to 640X-fold, correlating with the differences found in the promoter versions. MAIN CONCLUSIONSTherefore, the present study highlighted the link between the ST toxin gene and the superintegron, not only for V. cholerae, but also for V. mimicus. The differential st expression revealed that it is detrimental to the cognate promoter version, which would impact the toxin production and strain virulence.

genomics↗

bioRxiv2024

Genomic insights on the potential role of the accessory genome in the emergence of a novel geographically restricted K. pneumoniae lineage as a high-risk clone

Klebsiella pneumoniae causes life-threatening nosocomial infections and is featured by a remarkable propensity for multidrug resistance acquisition. Infections caused by multidrug- (MDR) and extensively drug-resistant (XDR) strains lead to a limitation of therapeutic options and an increase in persistent infections, and they are usually represented by high-risk lineages. Based on these features and their relevance to global public health, most of the studies focused on such high-risk clones, and little is known about the epidemiological and evolutionary dynamics of new/geographically restricted lineages. This study aimed to unveil the antimicrobial resistance and virulence genetic repertoire of a clinical XDR K. pneumoniae (Kp199) strain belonging to geographically-restricted ST, not linked to any known clonal complex. Its intrinsic (gyrA, parC, ramR, soxR and soxS mutations) and acquired resistome agreed with the observed XDR phenotype. An extensive arsenal of both antibiotic and heavy metal resistance genes was observed, as well as genes involved with resistance to several antiseptics currently used in clinical settings. The co-occurrence of blaKPC-2 and blaNDM-1 carbapenemase genes in Kp199 was an alarming finding since it could contribute to increased carbapenem resistance. Kp199 virulome was associated with bacterial survival and replication during infections. This study raises concern about a novel, geographically restricted K. pneumoniae lineage harbouring a huge resistome and virulome, which may strongly contribute to its successful establishment as an epidemic lineage. Therefore, our findings underscore the importance of vigilant surveillance and control measures to mitigate the threat posed by the potential emergence of new high-risk pandemic clones.

microbiology↗

bioRxiv2023

Fucose is an essential feature in cryoprotective polysaccharides

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Our current investigation studied 26 natural polysaccharides as potential CPA, probing correlations between post-thaw metabolic viability (PTV) of cryopreserved Vero cells and monomeric composition. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18-35.7 mol%), uronic acids (UA) (13.5-26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. To deconvolute multiple variable effects, principal component analysis (PCA) coupled to K-means clustering was performed. Two major mechanisms of action explained PTV variability: a charge-dependent effect of contrasting charged uronic acid and neutral monomer compositions, and a MW-scaled charge-independent mechanism exclusively attributed to fucose. Ultimately, our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection, highlighting the potential of fucose-rich polyanionic polysaccharides.

cell biology↗

bioRxiv2023

In-depth analysis of Klebsiella aerogenes resistome, virulome and mobilome worldwide

Klebsiella aerogenes is an emergent pathogen associated with outbreaks of carbapenem-resistant strains. To date, studies focusing on K. aerogenes have been small-scale and/or geographically restricted. Here, we analyzed the epidemiology, resistome, virulome, and mobilome of this species based on 561 genomes, spanning all continents. Furthermore, we sequenced four new strains from Brazil (mostly from the Amazon region). Dozens of STs occur worldwide, but the pandemic clones ST93 and ST4 have prevailed in several countries. Almost all genomes were clinical, however, most of them did not carry ESBL or carbapenemases, instead, they carried chromosomal alterations (omp36, ampD, ampG, ampR) associated with resistance to {beta}-lactams. Integrons were also identified, presenting gene cassettes not yet reported in this species (blaIMP, blaVIM, blaGES). Considering the virulence loci, the yersiniabactin and colibactin operons were found in the ICEKp10 element, which is disseminated in genomes of several STs, as well as an incomplete salmochelin cluster. In contrast, the aerobactin hypervirulence trait was observed only in one ST432 genome. Plasmids were common, mainly from the ColRNAI replicon, with some carrying resistance genes (mcr, blaTEM, blaNDM, blaIMP, blaKPC, blaVIM) and virulence genes (EAST1, senB). Interestingly, plasmids containing the colicin gene occurred in hundreds of genomes of different STs.

genomics↗

bioRxiv2023

Genomic characterization of a pandrug-resistant Klebsiella pneumoniae belonging to the high-risk ST11 in the Brazilian Amazon region

Pandrug-resistant (PDR) K. pneumoniae has been reported sporadically in many countries and remains rare in Brazil. The lack of genomic studies limits the comprehension of the determinants mostly involved with the PDR emergence in K. pneumoniae. This study aimed to unravel the main genetic determinants involved with the PDR background of a clinical ST11 K. pneumoniae recovered in the Brazilian Amazon region. The carbapenem-resistant Kp196 was submitted to WGS and its intrinsic and acquired resistome was assessed by CARD and comparison with wild-type genes. Kp196 resistome was composed of acquired resistance determinants and mutations in chromosomal genes. Among the formers, blaCTX-M-15 and blaNDM-1, blaOXA-9, blaOXA-1, aadA1, aacA4, strAB, aph(3)-VI, aac(3)-IId, qnrS1, qnrB1, oqxAB, dfrA14, sul2, catB3 were found in the vicinity of mobile genetic elements, which could contribute to their spread. Kp196 colistin resistance was multifactorial and attributed to modifications in ArnT (M114L/V117I/R372K), PhoQ (D150G), and the mgrB disruption by ISKpn25. Besides the presence of qnr and oqxAB genes, Kp196 also presented altered GyrA (S83I) and ParC (S80I). An in-block deletion in the repressor RamR, contributing to acrAB overexpression, and the presence of an enhanced-function AcrB variant (S966A), probably led to the Kp196 multidrug and tigecycline resistance. Insertions, in-block deletion, and missense mutations were involved with ompK35-36-37 inactivation, also accounting for the Kp196 multidrug resistance, including carbapenems. The Kp196 PDR profile, especially the carbapenem resistance, was due to the accumulation of different mechanisms, in which modifications in housekeeping genes accounted for a more stable resistome.

genomics↗

bioRxiv2021

Pericyte-mediated constriction of renal capillaries evokes no-reflow and kidney injury following ischemia

Acute kidney injury is common, with [~]13 million cases and 1.7 million deaths/year worldwide. A major cause is renal ischemia, typically following cardiac surgery, renal transplant or severe hemorrhage. We examined the cause of the sustained reduction in renal blood flow ("no-reflow"), which exacerbates kidney injury even after an initial cause of compromised blood supply is removed. After 60 min kidney ischemia and 30-60 min reperfusion, renal blood flow remained reduced, especially in the medulla, and kidney tubule damage was detected as Kim-1 expression. Constriction of the medullary descending vasa recta and cortical peritubular capillaries occurred near pericyte somata, and led to capillary blockages, yet glomerular arterioles and perfusion were unaffected, implying that the long-lasting decrease of renal blood flow contributing to kidney damage was generated by pericytes. Blocking Rho kinase to decrease pericyte contractility from the start of reperfusion increased the post-ischemic diameter of the descending vasa recta capillaries at pericytes, reduced the percentage of capillaries that remained blocked, increased medullary blood flow and reduced kidney injury. Thus, post-ischemic renal no-reflow, contributing to acute kidney injury, reflects pericytes constricting the descending vasa recta and peritubular capillaries. Pericytes are therefore an important therapeutic target for treating acute kidney injury.

pathology↗

bioRxiv2021

SARS-CoV-2 binding to ACE2 triggers pericyte-mediated angiotensin-evoked cerebral capillary constriction

The SARS-CoV-2 receptor, ACE2, is found on pericytes, contractile cells enwrapping capillaries that regulate brain, heart and kidney blood flow. ACE2 converts vasoconstricting angiotensin II into vasodilating angiotensin-(1-7). In brain slices from hamster, which has an ACE2 sequence similar to human ACE2, angiotensin II alone evoked only a small capillary constriction, but evoked a large pericyte-mediated capillary constriction generated by AT1 receptors in the presence of the SARS-CoV-2 receptor binding domain (RBD). The effect of the RBD was mimicked by blocking ACE2. A mutated non-binding RBD did not potentiate constriction. A similar RBD-potentiated capillary constriction occurred in human cortical slices. This constriction reflects an RBD-induced decrease in the conversion of angiotensin II to angiotensin-(1-7). The clinically-used drug losartan inhibited the RBD-potentiated constriction. Thus AT1 receptor blockers could be protective in SARS-CoV-2 infection by reducing pericyte-mediated blood flow reductions in the brain, and perhaps the heart and kidney.

neuroscience↗

bioRxiv2021

Emergence of XDR high-risk Pseudomonas aeruginosa ST309 in South America: a global comparative genomic analysis

Pseudomonas aeruginosa has been considered one of the major nosocomial pathogens associated with elevated morbidity and mortality worldwide. Outbreaks have been associated with few high-risk pandemic P. aeruginosa lineages, presenting a remarkable antimicrobial resistance. However, the biological features involved with the persistence and spread of such lineages among clinical settings remain to be unravel. This study reports the emergence of the ST309 P. aeruginosa lineage in South America/Brazil, more precisely, in the Amazon region. Global genomic analyses were performed with the Brazilian strain (PA834) and more 41 complete and draft ST309 genomes publicly available, giving insights about ST309 epidemiology and its resistome and mobilome. Antimicrobial susceptibility tests revealed that the Brazilian PA834 strain presented the XDR phenotype, which was mainly due to intrinsic resistance mechanisms. Genomic analyses revealed a heterogeneous distribution of acquired antimicrobial resistance genes among ST309 genomes, which included blaVIM-2, blaIMP-15 and qnrVC1, all of them associated with class 1 integrons. The mobilome mining showed the presence of Integrative and Conjugative Elements, transposons and genomic islands harbouring a huge arsenal of hevy metal resistance genes. Moreover, these elements also carried genes involved with virulence and adaptive traits. Therefore, the presence of such genes in ST309 lineage possibly accounted for the global spread and persistence of this emerging clone, and for its establishment as a pandemic lineage of clinical importance.

genomics↗

bioRxiv2019

Significant temporal shifts on clonal and plasmid backgrounds of Enterobacteriaceae producing acquired AmpC in Portuguese clinical settings

OBJECTIVESTo provide detailed molecular data on clinical acquired AmpC (qAmpC)-producing Enterobacteriaceae from two different periods (2002-2008 and 2010-2013) in order to clarify the contribution of clonal and plasmid genetic platforms for the current epidemiological scenario concerning extended-spectrum beta-lactams resistance. METHODSWe analysed 1246 Enterobacteriaceae non-susceptible to third-generation cephalosporins from 2 hospitals and 1 community laboratory between 2010 and 2013. Bacterial identification, antibiotic susceptibility, identification of qAmpC and plasmid-mediated quinolone resistance genes, clonal (PFGE, MLST) and plasmid (S1-/I-CeuI-PFGE, replicon typing, hybridization) analysis were performed by standard methods. WGS was performed in two ST11-K. pneumoniae isolates harbouring DHA-1. RESULTSThe occurrence of qAmpC was lower (2.6%) than that observed in a previous survey (7.4%), and varied slightly over time. Isolates produced DHA-1 (53%), CMY-2 (44%) or DHA-6 (3%), but significant epidemiological changes were observed in the two surveys. While DHA-1 persisted in different institutions by selection of a worldwide epidemic IncR plasmid in a ST11 harbouring KL105, CMY-2 rates increased over time linked to IncI1 plasmids (instead of IncK or IncA/C2) in multiple E. coli clones. CONCLUSIONSThe higher frequency of DHA-1 qAmpC in these species contrasts with the scenario of most European countries. Furthermore, the different genetic backgrounds associated with either ESBL or qAmpC in our country might have contributed to their differential expansion.

epidemiology↗