Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

In Vitro and In Vivo Biocompatibility of Natural and Synthetic Pseudomonas aeruginosa Pyomelanin for Potential Biomedical Applications.

Bacteria are the source of many bioactive compounds, including polymers with various physiological functions and the potential for medical applications. Pyomelanin from Pseudomonas aeruginosa, a nonfermenting Gram-negative bacterium, is a black-brown negatively charged extracellular polymer of homogentisic acid produced during L-tyrosine catabolism. Due to its chemical properties and the presence of active functional groups, pyomelanin is a candidate for the development of new antioxidant, antimicrobial and immunomodulatory formulations. This work aimed to obtain bacterial water-soluble (Pyosol), water-insoluble (Pyoinsol) and synthetic (sPyo) pyomelanin variants and characterize their chemical structure, thermosensitivity and biosafety in vitro and in vivo (Galleria mallonella). FTIR analysis showed that aromatic ring connections in the polymer chains were dominant in Pyosol and sPyo, whereas Pyoinsol had fewer Car-Car links between rings. The differences in chemical structure influence the solubility of various forms of pyomelanins, their thermal stability and biological activity. Pyosol and Pyoinsol showed higher biological safety than sPyo. The obtained results qualify Pyosol and Pyoinsol for evaluation of their antimicrobial, immunomodulatory and proregenerative activities.

Dual Modification of Porous Ca-P/PLA Composites with APTES and Alendronate Improves Their Mechanical Strength and Cytobiocompatibility towards Human Osteoblasts.

Synthetic implants are used to treat large bone defects that are often unable to regenerate, for example those caused by osteoporosis. It is necessary that the materials used to manufacture them are biocompatible and resorbable. Polymer-ceramic composites, such as those based on poly(L-lactide) (PLLA) and calcium phosphate ceramics (Ca-P), are often used for these purposes. In this study, we attempted to investigate an innovative strategy for two-step (dual) modification of composites and their components to improve the compatibility of composite components and the adhesion between PLA and Ca-P whiskers, and to increase the mechanical strength of the composite, as well as improve osteological bioactivity and prevent bone resorption in composites intended for bone regeneration. In the first step, Ca-P whiskers were modified with a saturated fatty acid namely, lauric acid (LA), or a silane coupling agent γ-aminopropyltriethoxysilane (APTES). Then, the composite, characterized by the best mechanical properties, was modified in the second stage of the work with an active chemical compound used in medicine as a first-line drug in osteoporosis-sodium alendronate, belonging to the group of bisphosphonates (BP). As a result of the research covered in this work, the composite modified with APTES and alendronate was found to be a promising candidate for future biomedical engineering applications.

Effects of Sterilization and Hydrolytic Degradation on the Structure, Morphology and Compressive Strength of Polylactide-Hydroxyapatite Composites.

Composites based on polylactide (PLA) and hydroxyapatite (HA) were prepared using a thermally induced phase separation method. In the experimental design, the PLA with low weight-average molar mass (Mw) and high Mw were tested with the inclusion of HA synthesized as whiskers or hexagonal rods. In addition, the structure of HA whiskers was doped with Zn, whereas hexagonal rods were mixed with Sr salt. The composites were sterilized and then incubated in phosphate-buffered saline for 12 weeks at 37 °C, followed by characterization of pore size distribution, molecular properties, density and mechanical strength. Results showed a substantial reduction of PLA Mw for both polymers due to the preparation of composites, their sterilization and incubation. The distribution of pore size effectively increased after the degradation process, whereas the sterilization, furthermore, had an impact on pore size distribution depending on HA added. The inclusion of HA reduced to some extent the degradation of PLA quantitatively in the weight loss in vitro compared to the control without HA. All produced materials showed no cytotoxicity when validated against L929 mouse skin fibroblasts and hFOB 1.19 human osteoblasts. The lack of cytotoxicity was accompanied by the immunocompatibility with human monocytic cells that were able to detect pyrogenic contaminants.

Proteomic and transcriptomic experiments reveal an essential role of RNA degradosome complexes in shaping the transcriptome of Mycobacterium tuberculosis.

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.

The Effect of Pore Size Distribution and l-Lysine Modified Apatite Whiskers (HAP) on Osteoblasts Response in PLLA/HAP Foam Scaffolds Obtained in the Thermally Induced Phase Separation Process.

In this research, we prepared foam scaffolds based on poly(l-lactide) (PLLA) and apatite whiskers (HAP) using thermally induced phase separation technique supported by the salt leaching process (TIPS-SL). Using sodium chloride having a size of (a) 150-315 µm, (b) 315-400 µm, and (c) 500-600 µm, three types of foams with different pore sizes have been obtained. Internal structure of the obtained materials has been investigated using SEM as well as µCT. The materials have been studied by means of porosity, density, and compression tests. As the most promising, the composite prepared with salt size of 500-600 µm was prepared also with the l-lysine modified apatite. The osteoblast hFOB 1.19 cell response for the scaffolds was also investigated by means of cell viability, proliferation, adhesion/penetration, and biomineralization. Direct contact cytotoxicity assay showed the cytocompatibility of the scaffolds. All types of foam scaffolds containing HAP whiskers, regardless the pore size or l-lysine modification induced significant stimulatory effect on the cal-cium deposits formation in osteoblasts. The PLLA/HAP scaffolds modified with l-lysine stimulated hFOB 1.19 osteoblasts proliferation. Compared to the scaffolds with smaller pores (150-315 µm and 315-400 µm), the PLLA/HAP foams with large pores (500-600 µm) promoted more effective ad-hesion of osteoblasts to the surface of the biomaterial.

Three Component Composite Scaffolds Based on PCL, Hydroxyapatite, and L-Lysine Obtained in TIPS-SL: Bioactive Material for Bone Tissue Engineering.

In this research, we describe the properties of three-component composite foam scaffolds based on poly(ε-caprolactone) (PCL) as a matrix and hydroxyapatite whiskers (HAP) and L-Lysine as fillers (PCL/HAP/Lys with wt% ratio 50/48/2). The scaffolds were prepared using a thermally induced phase separation technique supported by salt leaching (TIPS-SL). All materials were precisely characterized: porosity, density, water uptake, wettability, DSC, and TGA measurements and compression tests were carried out. The microstructure of the obtained scaffolds was analyzed via SEM. It was found that the PCL/HAP/Lys scaffold has a 45% higher Young's modulus and better wettability compared to the PCL/HAP system. At the same time, the porosity of the system was ~90%. The osteoblast hFOB 1.19 cell response was also investigated in osteogenic conditions (39 °C) and the cytokine release profile of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α was determined. Modification of PCL scaffolds with HAP and L-Lysine significantly improved the proliferation of pre-osteoblasts cultured on such materials.

PGS/HAp Microporous Composite Scaffold Obtained in the TIPS-TCL-SL Method: An Innovation for Bone Tissue Engineering.

In this research, we synthesize and characterize poly(glycerol sebacate) pre-polymer (pPGS) (1H NMR, FTiR, GPC, and TGA). Nano-hydroxyapatite (HAp) is synthesized using the wet precipitation method. Next, the materials are used to prepare a PGS-based composite with a 25 wt.% addition of HAp. Microporous composites are formed by means of thermally induced phase separation (TIPS) followed by thermal cross-linking (TCL) and salt leaching (SL). The manufactured microporous materials (PGS and PGS/HAp) are then subjected to imaging by means of SEM and µCT for the porous structure characterization. DSC, TGA, and water contact angle measurements are used for further evaluation of the materials. To assess the cytocompatibility and biological potential of PGS-based composites, preosteoblasts and differentiated hFOB 1.19 osteoblasts are employed as in vitro models. Apart from the cytocompatibility, the scaffolds supported cell adhesion and were readily populated by the hFOB1.19 preosteoblasts. HAp-facilitated scaffolds displayed osteoconductive properties, supporting the terminal differentiation of osteoblasts as indicated by the production of alkaline phosphatase, osteocalcin and osteopontin. Notably, the PGS/HAp scaffolds induced the production of significant amounts of osteoclastogenic cytokines: IL-1ß, IL-6 and TNF-α, which induced scaffold remodeling and promoted the reconstruction of bone tissue. Initial biocompatibility tests showed no signs of adverse effects of PGS-based scaffolds toward adult BALB/c mice.

Influence of Hydroxyapatite Surface Functionalization on Thermal and Biological Properties of Poly(l-Lactide)- and Poly(l-Lactide-co-Glycolide)-Based Composites.

Novel biocomposites of poly(L-lactide) (PLLA) and poly(l-lactide-co-glycolide) (PLLGA) with 10 wt.% of surface-modified hydroxyapatite particles, designed for applications in bone tissue engineering, are presented in this paper. The surface of hydroxyapatite (HAP) was modified with polyethylene glycol by using l-lysine as a linker molecule. The modification strategy fulfilled two important goals: improvement of the adhesion between the HAP surface and PLLA and PLLGA matrices, and enhancement of the osteological bioactivity of the composites. The surface modifications of HAP were confirmed by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), TGA, and elemental composition analysis. The influence of hydroxyapatite surface functionalization on the thermal and in vitro biological properties of PLLA- and PLLGA-based composites was investigated. Due to HAP modification with polyethylene glycol, the glass transition temperature of PLLA was reduced by about 24.5 °C, and melt and cold crystallization abilities were significantly improved. These achievements were scored based on respective shifting of onset of melt and cold crystallization temperatures and 1.6 times higher melt crystallization enthalpy compared with neat PLLA. The results showed that the surface-modified HAP particles were multifunctional and can act as nucleating agents, plasticizers, and bioactive moieties. Moreover, due to the presented surface modification of HAP, the crystallinity degree of PLLA and PLLGA and the polymorphic form of PLLA, the most important factors affecting mechanical properties and degradation behaviors, can be controlled.

Molecular basis for DNA strand displacement by NHEJ repair polymerases.

The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal (Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair.

Mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin.

Tuberculosis remains a major health concern worldwide. Eradication of its causative agent, the bacterial pathogen Mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. Despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. Hypoxic conditions have been widely used as an in vitro system to study this dormancy. Such studies identified a rearrangement of central carbon metabolism to exploit fermentative processes caused by the lack of oxygen. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is the enzyme at the center of these metabolic rearrangements. Although Pck is associated with gluconeogenesis under standard growth conditions, the enzyme can catalyze the reverse reaction, supporting anaplerosis of the tricarboxylic acid cycle, under conditions leading to slowed or stopped bacterial replication. To study the mechanisms that regulate the switch between two Pck functions, we systematically investigated factors influencing the gluconeogenic and anaplerotic reaction kinetics. We demonstrate that a reducing environment, as found under hypoxia-triggered non-replicating conditions, accelerates the reaction in the anaplerotic direction. Furthermore, we identified proteins that interact with Pck. The interaction between Pck and the reduced form of mycobacterial thioredoxin, gene expression of which is increased under hypoxic conditions, also increased the Pck anaplerotic activity. We thus propose that a reducing environment and the protein-protein interaction with thioredoxin in particular enable the Pck anaplerotic function under fermentative growth conditions.

Septal localization of the Mycobacterium tuberculosis MtrB sensor kinase promotes MtrA regulon expression.

The mechanisms responsible for activation of the MtrAB two-component regulatory signal transduction system, which includes sensor kinase MtrB and response regulator MtrA, are unknown. Here, we show that an MtrB-GFP fusion protein localized to the cell membrane, the septa, and the poles in Mycobacterium tuberculosis and Mycobacterium smegmatis. This localization was independent of MtrB phosphorylation status but dependent upon the assembly of FtsZ, the initiator of cell division. The M. smegmatis mtrB mutant was filamentous, defective for cell division, and contained lysozyme-sensitive cell walls. The mtrB phenotype was complemented by either production of MtrB protein competent for phosphorylation or overproduction of MtrA(Y102C) and MtrA(D13A) mutant proteins exhibiting altered phosphorylation potential, indicating that either MtrB phosphorylation or MtrB independent expression of MtrA regulon genes, including those involved in cell wall processing, are necessary for regulated cell division. In partial support of this observation, we found that the essential cell wall hydrolase ripA is an MtrA target and that the expression of bona fide MtrA targets ripA, fbpB, and dnaA were compromised in the mtrB mutant and partially rescued upon MtrA(Y102C) and MtrA(D13A) overproduction. MtrB septal assembly was compromised upon FtsZ depletion and exposure of cells to mitomycin C, a DNA damaging agent, which interferes with FtsZ ring assembly. Expression of MtrA targets was also compromised under the above conditions, indicating that MtrB septal localization and MtrA regulon expression are linked. We propose that MtrB septal association is a necessary feature of MtrB activation that promotes MtrA phosphorylation and MtrA regulon expression.

Depletion of tRNA CCA-adding enzyme in Mycobacterium tuberculosis leads to polyadenylation of transcripts and precursor tRNAs.

In reference to gene annotation, more than half of the tRNA species synthesized by Mycobacterium tuberculosis require the enzymatic addition of the cytosine-cytosine-adenine (CCA) tail, which is indispensable for amino acid charging and tRNA functionality. It makes the mycobacterial CCA-adding enzyme essential for survival of the bacterium and a potential target for novel pipelines in drug discovery avenues. Here, we described the rv3907c gene product, originally annotated as poly(A)polymerase (rv3907c, PcnA) as a functional CCA-adding enzyme (CCAMtb) essential for viability of M. tuberculosis. The depletion of the enzyme affected tRNAs maturation, inhibited bacilli growth, and resulted in abundant accumulation of polyadenylated RNAs. We determined the enzymatic activities displayed by the mycobacterial CCAMtb in vitro and studied the effects of inhibiting of its transcription in bacterial cells. We are the first to properly confirm the existence of RNA polyadenylation in mycobacteria, a previously controversial phenomenon, which we found promoted upon CCA-adding enzyme downexpression.

The functional response of human monocyte-derived macrophages to serum amyloid A and Mycobacterium tuberculosis infection.

Introduction: In the course of tuberculosis (TB), the level of major acute phase protein, namely serum amyloid A (hSAA-1), increases up to a hundredfold in the pleural fluids of infected individuals. Tubercle bacilli infecting the human host can be opsonized by hSAA-1, which affects bacterial entry into human macrophages and their intracellular multiplication. Methods: We applied global RNA sequencing to evaluate the functional response of human monocyte-derived macrophages (MDMs), isolated from healthy blood donors, under elevated hSAA-1 conditions and during infection with nonopsonized and hSAA-1-opsonized Mycobacterium tuberculosis (Mtb). In the same infection model, we also examined the functional response of mycobacteria to the intracellular environment of macrophages in the presence and absence of hSAA-1. The RNASeq analysis was validated using qPCR. The functional response of MDMs to hSAA-1 and/or tubercle bacilli was also evaluated for selected cytokines at the protein level by applying the Milliplex system. Findings: Transcriptomes of MDMs cultured in the presence of hSAA-1 or infected with Mtb showed a high degree of similarity for both upregulated and downregulated genes involved mainly in processes related to cell division and immune response, respectively. Among the most induced genes, across both hSAA-1 and Mtb infection conditions, CXCL8, CCL15, CCL5, IL-1ß, and receptors for IL-7 and IL-2 were identified. We also observed the same pattern of upregulated pro-inflammatory cytokines (TNFα, IL-6, IL-12, IL-18, IL-23, and IL-1) and downregulated anti-inflammatory cytokines (IL-10, TGFß, and antimicrobial peptide cathelicidin) in the hSAA-1 treated-MDMs or the phagocytes infected with tubercle bacilli. At this early stage of infection, Mtb genes affected by the inside microenvironment of MDMs are strictly involved in iron scavenging, adaptation to hypoxia, low pH, and increasing levels of CO2. The genes for the synthesis and transport of virulence lipids, but not cholesterol/fatty acid degradation, were also upregulated. Conclusion: Elevated serum hSAA-1 levels in tuberculosis enhance the response of host phagocytes to infection, including macrophages that have not yet been in contact with mycobacteria. SAA induces antigen processing and presentation processes by professional phagocytes reversing the inhibition caused by Mtb infection.

Bioactive Materials for Bone Regeneration: Biomolecules and Delivery Systems.

Novel tissue regeneration strategies are constantly being developed worldwide. Research on bone regeneration is noteworthy, as many promising new approaches have been documented with novel strategies currently under investigation. Innovative biomaterials that allow the coordinated and well-controlled repair of bone fractures and bone loss are being designed to reduce the need for autologous or allogeneic bone grafts eventually. The current engineering technologies permit the construction of synthetic, complex, biomimetic biomaterials with properties nearly as good as those of natural bone with good biocompatibility. To ensure that all these requirements meet, bioactive molecules are coupled to structural scaffolding constituents to form a final product with the desired physical, chemical, and biological properties. Bioactive molecules that have been used to promote bone regeneration include protein growth factors, peptides, amino acids, hormones, lipids, and flavonoids. Various strategies have been adapted to investigate the coupling of bioactive molecules with scaffolding materials to sustain activity and allow controlled release. The current manuscript is a thorough survey of the strategies that have been exploited for the delivery of biomolecules for bone regeneration purposes, from choosing the bioactive molecule to selecting the optimal strategy to synthesize the scaffold and assessing the advantages and disadvantages of various delivery strategies.

Calcination and ion substitution improve physicochemical and biological properties of nanohydroxyapatite for bone tissue engineering applications.

Nanohydroxyapatite (nanoHAP) is widely used in bone regeneration, but there is a need to enhance its properties to provide stimuli for cell commitment and osteoconduction. This study examines the effect of calcination at 1200 °C on the physicochemical and biological properties of nanoHAP doped with magnesium (Mg2+), strontium (Sr2+), and zinc (Zn2+). A synergistic effect of dual modification on nanoHAP biological properties was investigated. The materials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), BET analysis, Fourier-transform spectroscopy, and thermal analysis methods. Furthermore, ion release tests and in vitro biological characterization, including cytocompatibility, reactive oxygen species production, osteoconductive potential and cell proliferation, were performed. The XRD results indicate that the ion substitution of nanoHAP has no effect on the apatite structure, and after calcination, ß-tricalcium phosphate (ß-TCP) is formed as an additional phase. SEM analysis showed that calcination induces the agglomeration of particles and changes in surface morphology. A decrease in the specific surface area and in the ion release rate was observed. Combining calcination and nanoHAP ion modification is beneficial for cell proliferation and osteoblast response and provide additional stimuli for cell commitment in bone regeneration.

The Orphan Response Regulator Rv3143 Modulates the Activity of the NADH Dehydrogenase Complex (Nuo) in Mycobacterium tuberculosis via Protein-Protein Interactions.

Two-component signal transduction systems enable mycobacterial cells to quickly adapt and adequately respond to adverse environmental conditions encountered at various stages of host infection. We attempted to determine the role of the Rv3143 "orphan" response regulator in the physiology of Mycobacterium tuberculosis and its orthologue Msmeg_2064 in Mycobacterium smegmatis. We identified the Rv3143 protein as an interaction partner for NuoD, a member of the type I NADH dehydrogenase complex involved in oxidative phosphorylation. The mutants Δrv3143 and Δmsmeg_2064 were engineered in M. tuberculosis and M. smegmatis cells, respectively. The Δmsmeg_2064 strain exhibited a significant reduction in growth and viability in the presence of reactive nitrogen species. The Rv3143-deficient strain was sensitive to valinomycin, which is known to reduce the electrochemical potential of the cell and overexpressed genes required for nitrate respiration. An increased level of reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) electron acceptor in Δrv3143 and Δmsmeg_2064 cells was also evident. The silencing of ndh expression using CRISPRi/dCas9 affected cell survival under limited oxygen conditions. Oxygen consumption during entry to hypoxia was most severely affected in the double-mutant Δmsmeg_2064 ndhCRISPRi/dCas9 . We propose that the regulatory protein Rv3143 is a component of the Nuo complex and modulates its activity.

Small RNAs Asserting Big Roles in Mycobacteria.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells, and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50 and 350 nts in length and smaller RNAs (sncRNA) < 50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists, and locked nucleic acid sequence-specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions remaining about the biological and pathogenic roles of these small non-coding RNAs, and potential research directions needed to define the role of these mycobacterial noncoding RNAs are summarized.

Ten-eleven translocation methylcytosine dioxygenase 3-loaded microspheres penetrate neurons in vitro causing active demethylation and neurite outgrowth.

Epigenetic processes, such as DNA methylation and other chromatin modifications, are believed to be largely responsible for establishing a reduced capacity for growth in the mature nervous system. Ten-eleven translocation methylcytosine dioxygenase 3 (Tet3)-, a member of the Tet gene family, plays a crucial role in promoting injury-induced DNA demethylation and expression of regeneration-associated genes in the peripheral nervous system. Here, we encapsulate Tet3 protein within a clinically tolerated poly(lactide-co-glycolide) microsphere system. Next, we show that Tet3-loaded microspheres are internalized into mHippoE-18 embryonic hippocampal cells. We compare the outgrowth potential of Tet3 microspheres with that of commonly used nerve growth factor (NGF)-loaded microspheres in an in vitro injury model. Tet3-containing microspheres increased levels of nuclear 5-hydroxymethylcytosine indicating active demethylation and outperformed NGF-containing microspheres in measures of neurite outgrowth. Our results suggest that encapsulated demethylases may represent a novel avenue to treat nerve injuries.

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses.

Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.