Stochastic parabolic growth promotes coexistence and a relaxed error threshold in RNA-like replicator populations.

The RNA world hypothesis proposes that during the early evolution of life, primordial genomes of the first self-propagating evolutionary units existed in the form of RNA-like polymers. Autonomous, non-enzymatic, and sustained replication of such information carriers presents a problem, because product formation and hybridization between template and copy strands reduces replication speed. Kinetics of growth is then parabolic with the benefit of entailing competitive coexistence, thereby maintaining diversity. Here, we test the information-maintaining ability of parabolic growth in stochastic multispecies population models under the constraints of constant total population size and chemostat conditions. We find that large population sizes and small differences in the replication rates favor the stable coexistence of the vast majority of replicator species ('genes'), while the error threshold problem is alleviated relative to exponential amplification. In addition, sequence properties (GC content) and the strength of resource competition mediated by the rate of resource inflow determine the number of coexisting variants, suggesting that fluctuations in building block availability favored repeated cycles of exploration and exploitation. Stochastic parabolic growth could thus have played a pivotal role in preserving viable sequences generated by random abiotic synthesis and providing diverse genetic raw material to the early evolution of functional ribozymes.

All living things use molecules known as nucleic acids to store instructions on how to grow and maintain themselves and pass these instructions down to the next generation. However, it remains unclear how these systems may have evolved from simple molecules in the environment when life began over 3.6 billion years ago. One idea proposes that, before the first cells evolved, abiotic chemical processes gave rise to substantial building blocks of ribonucleic acids (or RNAs, for short). Over time, RNAs could have combined to form polymers of random sequences that started to copy themselves to make simple machines, only carrying the information required to make more of the same RNAs. Later on, these RNA molecules teamed up with proteins, fats and other molecules to make the first cells. When RNA replicates, the parent molecule is used as a template to assemble a new copy. While the new RNA molecule remains attached to its template it prevents the template being used to make more RNA. Therefore, it is thought that the speed at which a specific RNA machine copied itself may have varied in a pattern known as parabolic growth. Furthermore, when RNA replicates without the help of other biological molecules, the process is very prone to errors, which would have severely limited how much information the RNA machines were able to pass on to the next generation. Theoretical work suggested that under certain conditions, parabolic growth may favor the maintenance of a large amount of RNA sequence-coded information, but it is not clear if this is actually possible in nature. To address this question, Paczkó et al. developed mathematical models to investigate the effect of parabolic growth on the ability of RNA to replicate without other biological molecules. The models show that when large numbers of RNAs are present, small differences in how quickly different RNAs replicated favored the stable coexistence of different RNA sequences. Parabolic growth decreased the adverse effect of copying errors, allowing larger pieces of RNA to faithfully replicate themselves. This work suggests that parabolic growth may help to maintain different types of RNA (or similar replicating molecules) in a population and in turn, help new simple life forms to evolve. In the future, these findings may be used as a framework for laboratory experiments to better understand how early life forms may have evolved.

A neural network-based model framework for cell-fate decisions and development.

Gene regulatory networks (GRNs) fulfill the essential function of maintaining the stability of cellular differentiation states by sustaining lineage-specific gene expression, while driving the progression of development. However, accounting for the relative stability of intermediate differentiation stages and their divergent trajectories remains a major challenge for models of developmental biology. Here, we develop an empirical data-based associative GRN model (AGRN) in which regulatory networks store multilineage stage-specific gene expression profiles as associative memory patterns. These networks are capable of responding to multiple instructive signals and, depending on signal timing and identity, can dynamically drive the differentiation of multipotent cells toward different cell state attractors. The AGRN dynamics can thus generate diverse lineage-committed cell populations in a robust yet flexible manner, providing an attractor-based explanation for signal-driven cell fate decisions during differentiation and offering a readily generalizable modelling tool that can be applied to a wide variety of cell specification systems.

Decreased Expression of Placental Proteins in Recurrent Pregnancy Loss: Functional Relevance and Diagnostic Value.

Miscarriages affect 50-70% of all conceptions and 15-20% of clinically recognized pregnancies. Recurrent pregnancy loss (RPL, ≥2 miscarriages) affects 1-5% of recognized pregnancies. Nevertheless, our knowledge about the etiologies and pathophysiology of RPL is incomplete, and thus, reliable diagnostic/preventive tools are not yet available. Here, we aimed to define the diagnostic value of three placental proteins for RPL: human chorionic gonadotropin free beta-subunit (free-ß-hCG), pregnancy-associated plasma protein-A (PAPP-A), and placental growth factor (PlGF). Blood samples were collected from women with RPL (n = 14) and controls undergoing elective termination of pregnancy (n = 30) at the time of surgery. Maternal serum protein concentrations were measured by BRAHMS KRYPTOR Analyzer. Daily multiple of median (dMoM) values were calculated for gestational age-specific normalization. To obtain classifiers, logistic regression analysis was performed, and ROC curves were calculated. There were differences in changes of maternal serum protein concentrations with advancing healthy gestation. Between 6 and 13 weeks, women with RPL had lower concentrations and dMoMs of free ß-hCG, PAPP-A, and PlGF than controls. PAPP-A dMoM had the best discriminative properties (AUC = 0.880). Between 9 and 13 weeks, discriminative properties of all protein dMoMs were excellent (free ß-hCG: AUC = 0.975; PAPP-A: AUC = 0.998; PlGF: AUC = 0.924). In conclusion, free-ß-hCG and PAPP-A are valuable biomarkers for RPL, especially between 9 and 13 weeks. Their decreased concentrations indicate the deterioration of placental functions, while lower PlGF levels indicate problems with placental angiogenesis after 9 weeks.

Classification of preeclampsia according to molecular clusters with the goal of achieving personalized prevention.

The prevention of pre-eclampsia is difficult due to the syndromic nature and multiple underlying mechanisms of this severe complication of pregnancy. The current clinical distinction between early- and late-onset disease, although clinically useful, does not reflect the true nature and complexity of the pathologic processes leading to pre-eclampsia. The current gaps in knowledge on the heterogeneous molecular pathways of this syndrome and the lack of adequate, specific diagnostic methods are major obstacles to early screening and tailored preventive strategies. The development of novel diagnostic tools for detecting the activation of the identified disease pathways would enable early, accurate screening and personalized preventive therapies. We implemented a holistic approach that includes the utilization of different proteomic profiling methods of maternal plasma samples collected from various ethnic populations and the application of systems biology analysis to plasma proteomic, maternal demographic, clinical characteristic, and placental histopathologic data. This approach enabled the identification of four molecular subclasses of pre-eclampsia in which distinct and shared disease mechanisms are activated. The current review summarizes the results and conclusions from these studies and the research and clinical implications of our findings.

Coevolutionary stability of host-symbiont systems with mixed-mode transmission.

The coevolution of hosts and symbionts based on virulence and mode of transmission is a complex and diverse biological phenomenon. We introduced a conceptual model to study the stable coexistence and coevolution of an obligate symbiont (mutualist or parasite) with mixed-mode transmission and its host. Using an age-structured Leslie model for the host, we demonstrated how the obligate symbiont could modify the host's life history traits (survival and fecundity) and the long-term growth rate of the infected lineage. When the symbiont is vertically transmitted, we found that the host and its symbiont could maximize the infected lineage's evolutionary success (multi-level selection). Our model showed that symbionts' effect on host longevity and reproduction might differ, even be opposing, and their net effect might often be counterintuitive. The evolutionary stability of the ecologically stable coexistence was analyzed in the framework of coevolutionary dynamics. Moreover, we found conditions for the ecological and evolutionary stability of the resident host-symbiont pair, which does not allow invasion by rare mutants (each mutant dies out by ecological selection). We concluded that, within the context of our simplified model conditions, a host-symbiont system with mixed-mode transmission is evolutionarily stable unconditionally only if the host can maximize the Malthusian parameters of the infected and non-infected lineages using the same strategy. Finally, we performed a game-theoretical analysis of our selection situation and compared two stability definitions.

A Kinetic Transition Network Model Reveals the Diversity of Protein Dimer Formation Mechanisms.

Protein homodimers have been classified as three-state or two-state dimers depending on whether a folded monomer forms before association, but the details of the folding-binding mechanisms are poorly understood. Kinetic transition networks of conformational states have provided insight into the folding mechanisms of monomeric proteins, but extending such a network to two protein chains is challenging as all the relative positions and orientations of the chains need to be included, greatly increasing the number of degrees of freedom. Here, we present a simplification of the problem by grouping all states of the two chains into two layers: a dissociated and an associated layer. We combined our two-layer approach with the Wako-Saito-Muñoz-Eaton method and used Transition Path Theory to investigate the dimer formation kinetics of eight homodimers. The analysis reveals a remarkable diversity of dimer formation mechanisms. Induced folding, conformational selection, and rigid docking are often simultaneously at work, and their contribution depends on the protein concentration. Pre-folded structural elements are always present at the moment of association, and asymmetric binding mechanisms are common. Our two-layer network approach can be combined with various methods that generate discrete states, yielding new insights into the kinetics and pathways of flexible binding processes.

Directional selection coupled with kin selection favors the establishment of senescence.

BACKGROUND: Conventional wisdom in evolutionary theory considers aging as a non-selected byproduct of natural selection. Based on this, conviction aging was regarded as an inevitable phenomenon. It was also thought that in the wild organisms tend to die from diseases, predation and other accidents before they could reach the time when senescence takes its course. Evidence has accumulated, however, that aging is not inevitable and there are organisms that show negative aging even. Furthermore, old age does play a role in the deaths of many different organisms in the wild also. The hypothesis of programmed aging posits that a limited lifespan can evolve as an adaptation (i.e., positively selected for) in its own right, partly because it can enhance evolvability by eliminating "outdated" genotypes. A major shortcoming of this idea is that non-aging sexual individuals that fail to pay the demographic cost of aging would be able to steal good genes by recombination from aging ones. RESULTS: Here, we show by a spatially explicit, individual-based simulation model that aging can positively be selected for if a sufficient degree of kin selection complements directional selection. Under such conditions, senescence enhances evolvability because the rate of aging and the rate of recombination play complementary roles. The selected aging rate is highest at zero recombination (clonal reproduction). In our model, increasing extrinsic mortality favors evolved aging by making up free space, thereby decreasing competition and increasing drift, even when selection is stabilizing and the level of aging is set by mutation-selection balance. Importantly, higher extrinsic mortality is not a substitute for evolved aging under directional selection either. Reduction of relatedness decreases the evolved level of aging; chance relatedness favors non-aging genotypes. The applicability of our results depends on empirical values of directional and kin selection in the wild. CONCLUSIONS: We found that aging can positively be selected for in a spatially explicit population model when sufficiently strong directional and kin selection prevail, even if reproduction is sexual. The view that there is a conceptual link between giving up clonal reproduction and evolving an aging genotype is supported by computational results.

The interaction between mucin and poly(amino acid)s with controlled cationic group content in bulk phase and in thin layers.

The type and concentration of charged groups in polymers have a key role in mucoadhesive interactions. A series of cationic poly(amino acid)s with different charge densities was designed to unravel the correlation between chemical structure and mucin-polymer interactions. Colloidal interactions between the mucin protein and synthetic polyaspartamides were tested by dynamic light scattering, zeta potential measurements and turbidimetric titration as a function of polymer-to-mucin mass ratio. The mucoadhesive interactions displayed a strongly non-linear change with polymer composition. The attractive interactions between mucin and the polyaspartamides with at least 50 % cationic groups caused increased light scattering of dispersions due to the aggregation of mucin particles upon their charge reversal. Interactions were further analysed in a thin mucin layer to model life-like situations using a quartz crystal microbalance (QCM) in flow mode. Results pointed out that the fully cationic polyaspartamide is not necessarily superior to derivatives with lower cationic group content. The maximum of adsorbed mass of polymers on mucin was experienced at medium cationic group contents. This emphasizes the relevance of cationic polyaspartamides as mucoadhesive excipients due to their multiple functionalities and the possibility of fine-tuning their interactions with mucin via straightforward chemical steps.

Validation of the modified Alvarado score on patients attending A&E units with suspected appendicitis.

INTRODUCTION: The aim of our prospective study was to confirm validity and diagnostic accuracy of the modified Alvarado score, which was developed at the Department of Surgery, University of Szeged, on patients presenting with symptoms suggestive of acute appendicitis (right lower quadrant complaints) at the A&E department. PATIENT POPULATION, METHODS: 138 patients were included in our study between 01.01.2019 and 01.01.2020. For patients attending A&E, the first medic calculated and recorded the modified Alvarado score before surgical consultation. The consulting surgeon decided on further treatment without knowing the score. Validation of the score was based on the pathology report of the removed appendix (whether the operation was warranted, and if the score also supported indication for surgery), if there was readmission or surgery due to worsening symptoms after discharge from A&E. We also examined if there was any connection between the value of the Alvarado score and the severity of inflammation. Our aim was to prove that using modified Alvarado score at the A&E Units helps to reduce patient's waiting time and avoid unnecessary surgical consultations. Furthermore our study included measuring the diagnostic accuracy of the ultrasound examination (specificity, sensitivity). RESULTS: Based on the results, patients presenting at A&E had a mean modified Alvarado score of 6.5. Comparing the score to histological results showed that the specificity of the modified Alvarado score was 100%, and its sensitivity was 80.7%. Based on Spearman's rank correlation (0.796) and ROC analysis (AUC 0.968), the modified Alvarado score has an excellent predictive value in diagnosing acute appendicitis. When comparing the patients' waiting times with the use of modified Alvarado score and without it we found that there was a significant difference in group also in group under 4 points and in group over 7 points when using modified Alvarado score, so the diagnostic and therapeutic algorithm should be much quicker with the help of the score. We found a correlation between the severity of inflammation based on the Fisher's exact test. Rank correlation of the same question also showed a significant connection. All patients had an US examination during their diagnostic course, its sensitivity was 82.6%, specificity was 87%. Based on this, we can conclude that the predictive value of the imaging method is good. CONCLUSIONS: We can conclude according to our results that the predictive value of the modified score is excellent, and it can be safely applied by non-surgeons in urgent care in the differential diagnosis of acute appendicitis. The new score incorporates the results of an easily obtainable, ionising radiation free imaging method, the ultrasound, which was not included in previous scores. With the help of the new score, the number of unnecessary surgical referrals and waiting times for patients are reduced, excess examinations will become avoidable.

Beta-Secretase 1 Recruits Amyloid-Beta Precursor Protein to ROCK2 Kinase, Resulting in Erroneous Phosphorylation and Beta-Amyloid Plaque Formation.

The amyloidogenic processing of APP depends on two events: its phosphorylation by ROCK2 (at Thr654) and the phosphorylation of the APP-cleaving enzyme BACE1 (at Ser498). However, the mechanisms and structural details of APP-ROCK2 and BACE1-ROCK2 binding are unknown. Using direct physical methods in combination with an in silico approach, we found that BACE1 binds into the substrate-binding groove of ROCK2 with a low affinity (Kd = 18 µM), while no binding of APP to ROCK2 alone could be detected. On the other hand, a strong association (Kd = 3.5 nM) of APP to the weak ROCK2-BACE1 complex was observed, although no stable ternary complex was detected, i.e., BACE1 was displaced by APP. We constructed a sequential functional model: (1) BACE1 weakly binds to ROCK2 and induces an allosteric conformational change in ROCK2; (2) APP strongly binds to the ROCK2-BACE1 complex, and BACE1 is released; and (3) ROCK2 phosphorylates APP at Thr654 (leading to a longer stay in the early endosome during APP processing). Direct fluorescence titration experiments showed that the APP646-664 or APP665-695 fragments did not bind separately to the ROCK2-BACE1 complex. Based on these observations, we conclude that two binding sites are involved in the ROCK2-APP interaction: (1) the substrate-binding groove, where the APP646-664 sequence containing Thr654 sits and (2) the allosteric binding site, where the APP665-695 sequence binds. These results open the way to attack the allosteric site to prevent APP phosphorylation at Thr654 by ROCK2 without inhibiting the activity of ROCK2 towards its other substrates.

In situ gelation of thiolated poly(aspartic acid) derivatives through oxidant-free disulfide formation for ophthalmic drug delivery.

Efficient topical treatment of ocular diseases requires a prolonged residence time of drug formulations. An in situ gelling, mucoadhesive system can provide improved residence time while keeps the installation of the formulation easy and accurate due to its low initial viscosity. We synthesized a two-component, biocompatible water-based liquid formulation showing in situ gelation upon mixing. S-protected, preactivated derivatives of thiolated poly(aspartic acid) (PASP-SS-MNA) were synthesized by coupling the free thiol groups of thiolated poly(aspartic acid) (PASP-SH) with 6-mercaptonicotinic acid (MNA). The amount of protecting groups was 242, 341, and 530 µmol/g depending on the degree of thiolation of PASP. The chemical interaction between PASP-SS-MNA and mucin was proven, indicating the mucoadhesive properties. Disulfide cross-linked hydrogels were formed in situ without an oxidizing agent by mixing the aqueous solutions of PASP-SS-MNA and PASP-SH. The gelation time was controlled between 1 and 6 min, while the storage modulus was as high as 4-16 kPa depending on the composition. Swelling experiments showed that hydrogels with no residual thiol groups are stable in phosphate-buffered saline at pH = 7.4. In contrast, the presence of free thiol groups leads to the dissolution of the hydrogel with a rate depending on the excess of thiol groups. The biological safety of the polymers and MNA was confirmed on Madin-Darby Canine Kidney cell line. Furthermore, a prolonged release of ofloxacin was observed at pH = 7.4 compared to a conventional liquid formulation, supporting the potential of the developed biopolymers in ophthalmic drug delivery.

Evolutionary ecology of language origins through confrontational scavenging.

A dynamic model and an agent-based simulation model implementing the assumptions of the confrontational scavenging hypothesis on early protolanguage as an adaptive response of Homo erectus to gradual change in their habitat has been developed and studied. The core assumptions of the hypothesis and the model scenario are the pre-adaptation of our ancestors to occupy the ecological niche that they constructed for themselves by having evolved displaced communication and a rudimentary tool manufacture, two features allowing them to use a new, concentrated and abundant resource-megafauna carrion-on the savannahs replacing arboreal habitats owing to the drying climate of East Africa at about 2 Ma. The shift in diet required coordinated cooperation by the hominin scavengers confronted with concurrent predators. Power scavenging compelled displaced symbolic communication featuring a limited semantic range; syntax was not yet required. We show that phenotypic evolution on the accuracy of information transfer between cooperating hominins is a necessary and sufficient condition for the population of agents to survive the diet shift. Both the individual and the group fitness of the hominin horde increased with the accuracy of their protolanguage, with decreasing time allocated to foraging and thus more time left for culture. This article is part of the theme issue 'Human socio-cultural evolution in light of evolutionary transitions'.

Molecular subclasses of preeclampsia characterized by a longitudinal maternal proteomics study: distinct biomarkers, disease pathways and options for prevention.

OBJECTIVES: The heterogeneous nature of preeclampsia is a major obstacle to early screening and prevention, and a molecular taxonomy of disease is needed. We have previously identified four subclasses of preeclampsia based on first-trimester plasma proteomic profiles. Herein, we expanded this approach by using a more comprehensive panel of proteins profiled in longitudinal samples. METHODS: Proteomic data collected longitudinally from plasma samples of women who developed preeclampsia (n=109) and of controls (n=90) were available from our previous report on 1,125 proteins. Consensus clustering was performed to identify subgroups of patients with preeclampsia based on data from five gestational-age intervals by using select interval-specific features. Demographic, clinical, and proteomic differences among clusters were determined. Differentially abundant proteins were used to identify cluster-specific perturbed KEGG pathways. RESULTS: Four molecular clusters with different clinical phenotypes were discovered by longitudinal proteomic profiling. Cluster 1 involves metabolic and prothrombotic changes with high rates of early-onset preeclampsia and small-for-gestational-age neonates; Cluster 2 includes maternal anti-fetal rejection mechanisms and recurrent preeclampsia cases; Cluster 3 is associated with extracellular matrix regulation and comprises cases of mostly mild, late-onset preeclampsia; and Cluster 4 is characterized by angiogenic imbalance and a high prevalence of early-onset disease. CONCLUSIONS: This study is an independent validation and further refining of molecular subclasses of preeclampsia identified by a different proteomic platform and study population. The results lay the groundwork for novel diagnostic and personalized tools of prevention.

Thiolated cationic poly(aspartamides) with side group dependent gelation properties for the delivery of anionic polyelectrolytes.

In situ gellable polymers have potential applications as injectable formulations in drug delivery and regenerative medicine. Herein, thiolated cationic polyaspartamides were synthesised via two different approaches to correlate the side group structure with gelation properties, gel strength and drug release kinetics. Cysteamine (CEA) was used as a thiolating agent to prepare thiolated cationic polyaspartamide groups with short thiolated side groups. As a new pathway, thiolactone chemistry was integrated with cationic modification of polyaspartamides to prepare thiolated derivatives with longer, flexible side groups using N-acetyl-DL-homocysteine (NAH) thiolactone. Both types of thiolated polyaspartamides could be converted into stiff hydrogels under mild reaction conditions through oxidation-induced intermolecular disulfide formation. We confirmed that the longer side groups largely accelerated gelation and the stiffness of the resultant hydrogels was higher than that of the CEA-modified counterparts. Both the gelation time and stiffness could be adjusted by the degree of thiolation. Poly(aspartic acid) (PASP) derivatives with a controlled concentration of anionic groups were entrapped in the hydrogels during the in situ gelation. Based on the possible electrostatic interaction between the linear anionic polyelectrolytes and the cationic polymer network, we hypothesized that the release of the encapsulated material is controlled by the charge density. In accordance, fully anionic PASP was entrapped completely in the hydrogels, whereas a reduction in the number of anionic groups caused the partial release of PASP derivatives. NAH- and CEA- modified cationic polyaspartamide hydrogels showed distinct release rates, indicating the interplay between cationic and thiol functionalities in release kinetics.

Early pathways, biomarkers, and four distinct molecular subclasses of preeclampsia: The intersection of clinical, pathological, and high-dimensional biology studies.

Preeclampsia is a syndromic disease of the mother, fetus, and placenta. The main limitation in early and accurate diagnosis of preeclampsia is rooted in the heterogeneity of this syndrome as reflected by diverse molecular pathways, symptoms, and clinical outcomes. Gaps in our knowledge preclude successful early diagnosis, personalized treatment, and prevention. The advent of "omics" technologies and systems biology approaches addresses this problem by identifying the molecular pathways associated with the underlying mechanisms and clinical phenotypes of preeclampsia. Here, we provide a brief overview on how the field has progressed, focusing on studies utilizing state-of-the-art transcriptomics and proteomics methods. Moreover, we summarize our systems biology studies involving maternal blood proteomics and placental transcriptomics, which identified early maternal and placental disease pathways and showed that their interaction influences the clinical presentation of preeclampsia. We also present an analysis of maternal blood proteomics data which revealed distinct molecular subclasses of preeclampsia and their molecular mechanisms. Maternal and placental disease pathways behind these subclasses are similar to those recently reported in studies on the placental transcriptome. These findings may promote the development of novel diagnostic tools for the distinct subtypes of preeclampsia syndrome, enabling early detection and personalized follow-up and tailored care of patients.

Synthesis, complex formation and corneal permeation of cyclodextrin-modified, thiolated poly(aspartic acid) as self-gelling formulation of dexamethasone.

The present study aimed at developing a potential in situ gellable dexamethasone (DXM) eye drop. Poly(aspartic acid) (PASP) derivatives were synthesized with dual functionality to improve the solubility of DXM, and to achieve in situ gelation. First, amine-modified ß-cyclodextrin (CD) was attached to polysuccinimide (PSI), second, thiol functionalities were added by the reaction of cysteamine and succinimide rings. Finally, the PSI derivatives were hydrolysed to the corresponding PASP derivatives to get water-soluble polymers. Phase-solubility studies confirmed the complexation ability of CD-containing PASP derivatives. In situ gelation and the effect of the CD immobilization on this behaviour were characterized by rheological measurements. The solubilizing effect of CD was confirmed by kinetic solubility measurements, whereas in vitro corneal permeability assay (corneal-PAMPA) measurements were performed to determine in vitro permeability and flux values. The effect of the PASP derivatives on permeation strongly depended on chemical composition and polymer concentration.

A robust mucin-containing poly(vinyl alcohol) hydrogel model for the in vitro characterization of mucoadhesion of solid dosage forms.

Mucoadhesion testing at macroscopic scale needs a robust, convenient in vitro method as ex vivo methods suffer from poor reproducibility and ethical problems. Here we synthesized mucin-free poly(vinyl alcohol) (PVA) and mucin-containing PVA hydrogel substrates (Muc/PVA) to measure adhesion of polymer tablets. Freezing-thawing method was used for gelation to avoid chemical cross-linking and to preserve the functionality of mucin. The adhesion of first generation mucoadhesive polymers, poly(acrylic acid) (PAA) and hydroxypropylmethylcellulose (HPMC) was tested with outstanding reproducibility on individual batches of hydrogels and qualitative agreement with ex vivo literature data. Negatively charged PAA was less adhesive on Muc/PVA surface than on mucin-free PVA whereas HPMC as a neutral polymer displayed similar adhesion strength on both surfaces. Chitosan as a positively charged polymer showed enhanced adhesion on Muc/PVA substrate compared to mucin-free PVA. These results are corroborated by turbidimetric titration which indicated attractive electrostatic interactions between chitosan and mucin in contrast to the lack of attractive interactions for PAA and HPMC. These results prove the role of electronic theory in macroscopic mucoadhesion.

Cross-Linked Enzyme-Adhered Nanoparticles (CLEANs) for Continuous-Flow Bioproduction.

Nanostructured but micro-sized biocatalysts were created by bottom-up technology using multi-functionalized silica nanoparticles (NPs) as nano-sized building blocks to form cross-linked enzyme-adhered nanoparticles (CLEANs) as robust micro-sized particles with beneficial internal structure and good mechanical properties. Systematic surface modification of NPs with a grafting mixture consisting of organosilanes with reactive (aminopropyl) and inert (e. g., vinyl, propyl, phenyl, or octyl) functions resulted in functional NPs enabling cross-linking agents, such as glutardialdehyde or bisepoxides (glycerol diglycidyl ether, neopentylglycol diglycidyl ether, and poly(propylene glycol) diglycidyl ether), to bind and cross-link enzymes covalently and to form macroporous microparticles. These CLEANs were able to diminish several weaknesses of traditional cross-linked enzyme aggregates as biocatalysts, such as poor mechanical resistance, difficult recovery, and storage, strengthening their use for packed-bed enzyme reactors. Lipase B from Candida antarctica (CaLB) was selected as model enzyme for development of robust CLEANs, which were successfully tested for various industrially relevant applications including a kinetic resolution of a racemic alcohol and the production of various natural fragrance compounds under continuous-flow conditions.

Epigenetic Dysregulation of Trophoblastic Gene Expression in Gestational Trophoblastic Disease.

Gestational trophoblastic diseases (GTDs) have not been investigated for their epigenetic marks and consequent transcriptomic changes. Here, we analyzed genome-wide DNA methylation and transcriptome data to reveal the epigenetic basis of disease pathways that may lead to benign or malignant GTDs. RNA-Seq, mRNA microarray, and Human Methylation 450 BeadChip data from complete moles and choriocarcinoma cells were bioinformatically analyzed. Paraffin-embedded tissues from complete moles and control placentas were used for tissue microarray construction, DNMT3B immunostaining and immunoscoring. We found that DNA methylation increases with disease severity in GTDs. Differentially expressed genes are mainly upregulated in moles while predominantly downregulated in choriocarcinoma. DNA methylation principally influences the gene expression of villous trophoblast differentiation-related or predominantly placenta-expressed genes in moles and choriocarcinoma cells. Affected genes in these subsets shared focal adhesion and actin cytoskeleton pathways in moles and choriocarcinoma. In moles, cell cycle and differentiation regulatory pathways, essential for trophoblast/placental development, were enriched. In choriocarcinoma cells, hormone biosynthetic, extracellular matrix-related, hypoxic gene regulatory, and differentiation-related signaling pathways were enriched. In moles, we found slight upregulation of DNMT3B protein, a developmentally important de novo DNA methylase, which is strongly overexpressed in choriocarcinoma cells that may partly be responsible for the large DNA methylation differences. Our findings provide new insights into the shared and disparate molecular pathways of disease in GTDs and may help in designing new diagnostic and therapeutic tools.

Nanofibrous Formulation of Cyclodextrin Stabilized Lipases for Efficient Pancreatin Replacement Therapies.

Enzyme replacement therapies (ERT) have been of great help over the past 30 years in the treatment of various lysosomal storage disorders, including chronic pancreatitis and its common complication, exocrine pancreatic insufficiency. Research shows that difficulties in designing such drugs can be overcome by using appropriate additives and various enzyme immobilization techniques. Cyclodextrins (CDs) can be considered as a promising additive for enzyme replacement therapies, as they are known to enhance the activity of enzymes in a complex process due to their specific binding. In this study, we investigated the formulation of lipases (from Aspergillus oryzae and Burkholderia cepacia) paired with different cyclodextrins in poly(vinyl alcohol) (PVA) nanofibers by electrospinning technique. We examined the effect of the presence of cyclodextrins and nanoformulation on the lipase activity. The rheological and morphological characterizations of precursors and nanofibers were also performed using a viscometer as well as electron and Raman microscope. We found that by selecting the appropriate CD:lipase ratio, the activity of the investigated enzyme could be multiplied, and cyclodextrins can support the homogeneous dispersion of lipases inside the solid formula. In addition, the entrapment of lipases in PVA nanofibers led to a significant increase in activity compared to the preformulated precursor. In this way, the nanofibrous formulation of lipases combining CDs as additives can provide an efficient and sustainable possibility for designing novel solid medicines in ERT.