Variants of Oxidized Regenerated Cellulose and Their Distinct Effects on Neuronal Tissue

Based on oxidized regenerated cellulose (ORC), several hemostyptic materials, such as Tabotamp^®, Equicel^® and Equitamp^®, have been developed to approach challenging hemostasis in neurosurgery. The present study compares ORC that differ in terms of compositions and properties, regarding their structure, solubility, pH values and effects on neuronal tissue. Cytotoxicity was detected via DNA-binding fluorescence dye in Schwann cells, astrocytes, and neuronal cells. Additionally, organotypic hippocampal slice cultures (OHSC) were analyzed, using propidium iodide, hematoxylin-eosin, and isolectin B₄ staining to investigate the cellular damage, cytoarchitecture, and microglia activation. Whereas Equicel^® led to a neutral pH, Tabotamp^® (pH 2.8) and Equitamp^® (pH 4.8) caused a significant reduction of pH (p < 0.001). Equicel^® and Tabotamp^® increased cytotoxicity significantly in several cell lines (p < 0.01). On OHSC, Tabotamp^® and Equicel^® led to a stronger and deeper damage to the neuronal tissue than Equitamp^® or gauze (p < 0.01). Equicel^® increased strongly the number of microglia cells after 24 h (p < 0.001). Microglia cells were not detectable after Tabotamp^® treatment, presumably due to an artifact caused by strong pH reduction. In summary, our data imply the use of Equicel^®, Tabotamp^® or Equitamp^® for specific applications in distinct clinical settings depending on their localization or tissue properties.     

Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

Anorganic bovine bone matrix (Bio-Oss^®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss^®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss^® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were formed in a white rabbit model and then implanted or not (controls) with Bio-Oss^® or BMP-2/Bio-Oss^®. The Bio-Oss^® and BMP-2/Bio-Oss^® groups had significantly greater new bone areas (expressed as percentages of augmented areas) than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss^® group (16.50 ± 2.87 (n = 6)) had significantly greater new bone areas than the Bio-Oss^® group (9.43 ± 3.73 (n = 6)) at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss^® markedly enhances bone regeneration.     

Unique Regulation of Intestinal Villus Epithelial Cl−/HCO3− Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na⁺/H⁺ and Cl⁻/HCO₃⁻ exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl⁻/HCO₃⁻ exchange, measured as DIDS-sensitive ³⁶Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered K_m with no effects on V_max. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl⁻/HCO₃⁻ exchangers, were unaltered. Thus, inhibition of villus cell Cl⁻/HCO₃⁻ exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl⁻, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.     

Vitamin D Reverses Disruption of Gut Epithelial Barrier Function Caused by Campylobacter jejuni

Infections by the zoonotic foodborne bacterium Campylobacter jejuni (C. jejuni) are among the most frequent causes of bacterial gastroenteritis worldwide. The aim was to evaluate the relationship between epithelial barrier disruption, mucosal immune activation, and vitamin D (VD) treatment during C. jejuni infection, using intestinal epithelial cells and mouse models focused on the interaction of C. jejuni with the VD signaling pathway and VD treatment to improve C. jejuni-induced barrier dysfunction. Our RNA-Seq data from campylobacteriosis patients demonstrate inhibition of VD receptor (VDR) downstream targets, consistent with suppression of immune function. Barrier-preserving effects of VD addition were identified in C. jejuni-infected epithelial cells and IL-10^−/− mice. Furthermore, interference of C. jejuni with the VDR pathway was shown via VDR/retinoid X receptor (RXR) interaction. Paracellular leakiness of infected epithelia correlated with tight junction (TJ) protein redistribution off the TJ domain and apoptosis induction. Supplementation with VD reversed barrier impairment and prevented inhibition of the VDR pathway, as shown by restoration of transepithelial electrical resistance and fluorescein (332 Da) permeability. We conclude that VD treatment restores gut epithelial barrier functionality and decreases bacterial transmigration and might, therefore, be a promising compound for C. jejuni treatment in humans and animals.     

Human Recombinant DNase I (Pulmozyme®) Inhibits Lung Metastases in Murine Metastatic B16 Melanoma Model That Correlates with Restoration of the DNase Activity and the Decrease SINE/LINE and c-Myc Fragments in Blood Cell-Free DNA

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme^®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme^® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme^® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme^®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

Na+ and/or Cl− Toxicities Determine Salt Sensitivity in Soybean (Glycine max (L.) Merr.), Mungbean (Vigna radiata (L.) R. Wilczek), Cowpea (Vigna unguiculata (L.) Walp.), and Common Bean (Phaseolus vulgaris L.)

Grain legumes are important crops, but they are salt sensitive. This research dissected the responses of four (sub)tropical grain legumes to ionic components (Na⁺ and/or Cl⁻) of salt stress. Soybean, mungbean, cowpea, and common bean were subjected to NaCl, Na⁺ salts (without Cl⁻), Cl⁻ salts (without Na⁺), and a “high cation” negative control for 57 days. Growth, leaf gas exchange, and tissue ion concentrations were assessed at different growing stages. For soybean, NaCl and Na⁺ salts impaired seed dry mass (30% of control), more so than Cl⁻ salts (60% of control). All treatments impaired mungbean growth, with NaCl and Cl⁻ salt treatments affecting seed dry mass the most (2% of control). For cowpea, NaCl had the greatest adverse impact on seed dry mass (20% of control), while Na⁺ salts and Cl⁻ salts had similar intermediate effects (~45% of control). For common bean, NaCl had the greatest adverse effect on seed dry mass (4% of control), while Na⁺ salts and Cl⁻ salts impaired seed dry mass to a lesser extent (~45% of control). NaCl and Na⁺ salts (without Cl⁻) affected the photosynthesis (P_n) of soybean more than Cl⁻ salts (without Na⁺) (50% of control), while the reverse was true for mungbean. Na⁺ salts (without Cl⁻), Cl⁻ salts (without Na⁺), and NaCl had similar adverse effects on P_n of cowpea and common bean (~70% of control). In conclusion, salt sensitivity is predominantly determined by Na⁺ toxicity in soybean, Cl⁻ toxicity in mungbean, and both Na⁺ and Cl⁻ toxicity in cowpea and common bean.     

Cultivation of Keratinocytes and Fibroblasts in a Three-Dimensional Bovine Collagen-Elastin Matrix (Matriderm?) and Application for Full Thickness Wound Coverage in Vivo

New skin substitutes for burn medicine or reconstructive surgery pose an important issue in plastic surgery. Matriderm^® is a clinically approved three-dimensional bovine collagen-elastin matrix which is already used as a dermal substitute of full thickness burn wounds. The drawback of an avital matrix is the limited integration in full thickness skin defects, depending on the defect size. To further optimize this process, Matriderm^® has also been studied as a matrix for tissue engineering of skin albeit long-term cultivation of the matrix with cells has been difficult. Cells have generally been seeded onto the matrix with high cell loss and minimal time-consuming migration. Here we developed a cell seeded skin equivalent after microtransfer of cells directly into the matrix. First, cells were cultured, and microinjected into Matriderm^®. Then, cell viability in the matrix was determined by histology in vitro. As a next step, the skin substitute was applied in vivo into a full thickness rodent wound model. The wound coverage and healing was observed over a period of two weeks followed by histological examination assessing cell viability, proliferation and integration into the host. Viable and proliferating cells could be found throughout the entire matrix. The presented skin substitute resembles healthy skin in morphology and integrity. Based on this study, future investigations are planned to examine behaviour of epidermal stem cells injected into a collagen-elastin matrix under the aspects of establishment of stem cell niches and differentiation.     

Music-Based Intervention Ameliorates Mecp2-Loss-Mediated Sociability Repression in Mice through the Prefrontal Cortex FNDC5/BDNF Pathway

Patients with Rett syndrome (RTT) show severe difficulties with communication, social withdrawl, and learning. Music-based interventions improve social interaction, communication skills, eye contact, and physical skills and reduce seizure frequency in patients with RTT. This study aimed to investigate the mechanism by which music-based interventions compromise sociability impairments in mecp2 ^null/y mice as an experimental RTT model. Male mecp2 ^null/y mice and wild-type mice (24 days old) were randomly divided into control, noise, and music-based intervention groups. Mice were exposed to music or noise for 6 h/day for 3 consecutive weeks. Behavioral patterns, including anxiety, spontaneous exploration, and sociability, were characterized using open-field and three-chamber tests. BDNF, TrkB receptor motif, and FNDC5 expression in the prefrontal cortex (PFC), hippocampus, basal ganglia, and amygdala were probed using RT-PCR or immunoblotting. mecp2 ^null/y mice showed less locomotion in an open field than wild-type mice. The social novelty rather than the sociability of these animals increased following a music-based intervention, suggesting that music influenced the mecp2-deletion-induced social interaction repression rather than motor deficit. Mechanically, the loss of BDNF signaling in the prefrontal cortex and hippocampal regions, but not in the basal ganglia and amygdala, was compromised following the music-based intervention in mecp2 ^null/y mice, whereas TrkB signaling was not significantly changed in either region. FNDC5 expression in the prefrontal cortex region in mecp2 ^null/y mice also increased following the music-based intervention. Collective evidence reveals that music-based interventions improve mecp2-loss-induced social dysfunction. BDNF and FNDC5 signaling in the prefrontal cortex region mediates the music-based-intervention promotion of social interactions. This study gives new insight into the mechanisms underlying the improvement of social behaviors in mice suffering from experimental Rett syndrome following a music-based intervention.     

In Vitro Evaluation of No-Carrier-Added Radiolabeled Cisplatin ([189, 191Pt]cisplatin) Emitting Auger Electrons

Due to their short-range (2–500 nm), Auger electrons (Auger e⁻) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Auger e⁻, it remains challenging to maximize the interaction between Auger e⁻ and DNA. To assess the DNA-damaging effect of Auger e⁻ released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [^{189, 191}Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by immunofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incubation), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by the release of Auger e⁻ very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e⁻.     

Cystine/Glutamate Antiporter in Schizophrenia: From Molecular Mechanism to Novel Biomarker and Treatment

Glutamate, a crucial excitatory neurotransmitter, plays a major role in the modulation of schizophrenia’s pathogenesis. New drug developments for schizophrenia have been prompted by the hypoglutamatergic hypothesis of schizophrenia. The cystine/glutamate antiporter system x_c⁻ is related to glutamate-release regulation. Patients with schizophrenia were recently discovered to exhibit downregulation of x_c⁻ subunits—the solute carrier (SLC) family 3 member 2 and the SLC family 7 member 11. We searched for relevant studies from 1980, when Bannai and Kitamura first identified the protein subunit system x_c⁻ in lung fibroblasts, with the aim of compiling the biological, functional, and pharmacological characteristics of antiporter x_c⁻, which consists of several subunits. Some of them can significantly stimulate the human brain through the glutamate pathway. Initially, extracellular cysteine activates neuronal x_c⁻, causing glutamate efflux. Next, excitatory amino acid transporters enhance the unidirectional transportation of glutamate and sodium. These two biochemical pathways are also crucial to the production of glutathione, a protective agent for neural and glial cells and astrocytes. Investigation of the expression of system x_c⁻ genes in the peripheral white blood cells of patients with schizophrenia can facilitate better understanding of the mental disorder and future development of novel biomarkers and treatments for schizophrenia. In addition, the findings further support the hypoglutamatergic hypothesis of schizophrenia.     

Cloning,Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The K_m and V_max values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6), displaying optimal activity with p-nitrophenyl-C4 (K_cat/K_m = 11.74 mM⁻¹·S⁻¹). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.     

In Vitro Activity of Copper(II) Complexes,Loaded or Unloaded into a Nanostructured Lipid System,against Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis (Mtb), presenting 9.5 million new cases and 1.5 million deaths in 2014. The aim of this study was to evaluate a nanostructured lipid system (NLS) composed of 10% phase oil (cholesterol), 10% surfactant (soy phosphatidylcholine, sodium oleate), and Eumulgin^® HRE 40 ([castor oil polyoxyl-40-hydrogenated] in a proportion of 3:6:8), and an 80% aqueous phase (phosphate buffer pH = 7.4) as a tactic to enhance the in vitro anti-Mtb activity of the copper(II) complexes [CuCl₂(INH)₂]·H₂O (1), [Cu(NCS)₂(INH)₂]·5H₂O (2) and [Cu(NCO)₂(INH)₂]·4H₂O (3). The Cu(II) complex-loaded NLS displayed sizes ranging from 169.5 ± 0.7095 to 211.1 ± 0.8963 nm, polydispersity index (PDI) varying from 0.135 ± 0.0130 to 0.236 ± 0.00100, and zeta potential ranging from −0.00690 ± 0.0896 to −8.43 ± 1.63 mV. Rheological analysis showed that the formulations behave as non-Newtonian fluids of the pseudoplastic and viscoelastic type. Antimycobacterial activities of the free complexes and NLS-loaded complexes against Mtb H₃₇Rv ATCC 27294 were evaluated by the REMA methodology, and the selectivity index (SI) was calculated using the cytotoxicity index (IC₅₀) against Vero (ATCC^® CCL-81), J774A.1 (ATCC^® TIB-67), and MRC-5 (ATCC^® CCL-171) cell lines. The data suggest that the incorporation of the complexes into NLS improved the inhibitory action against Mtb by 52-, 27-, and 4.7-fold and the SI values by 173-, 43-, and 7-fold for the compounds 1, 2 and 3, respectively. The incorporation of the complexes 1, 2 and 3 into the NLS also resulted in a significant decrease of toxicity towards an alternative model (Artemia salina L.). These findings suggest that the NLS may be considered as a platform for incorporation of metallic complexes aimed at the treatment of TB.     

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera^® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera^® and Reditux™ displayed differences at the molecular level. MabThera^® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera^®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.     

Changing Microspatial Patterns of Sulfate-Reducing Microorganisms (SRM) during Cycling of Marine Stromatolite Mats

Microspatial arrangements of sulfate-reducing microorganisms (SRM) in surface microbial mats (~1.5 mm) forming open marine stromatolites were investigated. Previous research revealed three different mat types associated with these stromatolites, each with a unique petrographic signature. Here we focused on comparing “non-lithifying” (Type-1) and “lithifying” (Type-2) mats. Our results revealed three major trends: (1) Molecular typing using the dsrA probe revealed a shift in the SRM community composition between Type-1 and Type-2 mats. Fluorescence in-situ hybridization (FISH) coupled to confocal scanning-laser microscopy (CSLM)-based image analyses, and ³⁵SO₄ ²⁻-silver foil patterns showed that SRM were present in surfaces of both mat types, but in significantly (p < 0.05) higher abundances in Type-2 mats. Over 85% of SRM cells in the top 0.5 mm of Type-2 mats were contained in a dense 130 μm thick horizontal layer comprised of clusters of varying sizes; (2) Microspatial mapping revealed that locations of SRM and CaCO₃ precipitation were significantly correlated (p < 0.05); (3) Extracts from Type-2 mats contained acylhomoserine-lactones (C4-, C6-, oxo-C6 C7-, C8-, C10-, C12-, C14-AHLs) involved in cell-cell communication. Similar AHLs were produced by SRM mat-isolates. These trends suggest that development of a microspatially-organized SRM community is closely-associated with the hallmark transition of stromatolite surface mats from a non-lithifying to a lithifying state.     

Altered mRNA and Protein Expression of Monocarboxylate Transporter MCT1 in the Cerebral Cortex and Cerebellum of Prion Protein Knockout Mice

The effect of a cellular prion protein (PrP^c) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrP^c in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp^−/− mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na⁺/K⁺ ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp^−/− vs. WT control group, without a substantial change in the Na⁺/K⁺ ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp^−/− vs. WT mice. The present study demonstrates that a lack of PrP^c leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp^−/− vs. WT mice. Our findings provide evidence that PrP^c might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.     

Potential of Melt Electrowritten Scaffolds Seeded with Meniscus Cells and Mesenchymal Stromal Cells

Meniscus injury and meniscectomy are strongly related to osteoarthritis, thus there is a clinical need for meniscus replacement. The purpose of this study is to create a meniscus scaffold with micro-scale circumferential and radial fibres suitable for a one-stage cell-based treatment. Poly-caprolactone-based scaffolds with three different architectures were made using melt electrowriting (MEW) technology and their in vitro performance was compared with scaffolds made using fused-deposition modelling (FDM) and with the clinically used Collagen Meniscus Implants^® (CMI^®). The scaffolds were seeded with meniscus and mesenchymal stromal cells (MSCs) in fibrin gel and cultured for 28 d. A basal level of proteoglycan production was demonstrated in MEW scaffolds, the CMI^®, and fibrin gel control, yet within the FDM scaffolds less proteoglycan production was observed. Compressive properties were assessed under uniaxial confined compression after 1 and 28 d of culture. The MEW scaffolds showed a higher Young’s modulus when compared to the CMI^® scaffolds and a higher yield point compared to FDM scaffolds. This study demonstrates the feasibility of creating a wedge-shaped meniscus scaffold with MEW using medical-grade materials and seeding the scaffold with a clinically-feasible cell number and -type for potential translation as a one-stage treatment.     

A Comparative Study on the Biosorption of Cd2+ onto Paecilomyces lilacinus XLA and Mucoromycote sp. XLC

The filamentous fungi XLA and XLC isolated from Cd-contaminated soil were identified morphologically and phylogenetically as Paecilomyces lilacinus and Mucoromycote sp., respectively. The minimum inhibitory concentrations (MICs) of Cd²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cr³⁺ and Cr⁶⁺ in minimum mineral (MM) medium agar plates were 29,786, 2945, 9425, 5080, 1785 and 204 mg·L⁻¹ for XLA and 11,240, 884, 9100, 2540, 3060 and 51 mg·L⁻¹ for XLC, respectively. Favorable biosorption conditions for adsorption of Cd²⁺ by the tested fungi were investigated. Efficient performances of the biosorbents were described using Langmuir isotherm model, and the predicted maximum biosorption capacities for Cd²⁺ were 77.61 mg·g⁻¹ of XLA and 79.67 mg·g⁻¹of XLC. Experiments on desorption potential of biosorbents validated their efficacy at a large scale. Results showed that XLA obtained a desorption rate of 84.7% by 2% EDTA and XLC gained a desorption rate of 78.9% by 0.1 M HCl. Analysis by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) and X-ray photoelectron spectroscopy (XPS) suggested that groups of C–N, COO– for XLA and C–N, CH₂ and phosphate for XLC were the dominant binding sites for Cd²⁺ biosorption. Our results indicated that the fungus XLA, rather than XLC, could potentially be used as an inexpensive, eco-friendly and effective bioremediation agent for the removal of Cd²⁺ from wastewater.     

A High Redox Potential Laccase from Pycnoporus sanguineus RP15: Potential Application for Dye Decolorization

Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH₄Cl and 50 mmol·L⁻¹ CuSO₄, initial moisture 4.1 mL·g⁻¹), the laccase activity reached 138.6 ± 13.2 U·g⁻¹. Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0–8.0, and after two hours at 55–60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 μmol·L⁻¹, maximum velocity of 413.4 ± 21.2 U·mg⁻¹ and catalytic efficiency of 3140.1 ± 149.6 L·mmol⁻¹·s⁻¹. The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.     

BiP Heterozigosity Aggravates Pathological Deterioration in Experimental Amyotrophic Lateral Sclerosis

In the present study, we investigated the involvement of the chaperone protein BiP (also known as GRP78 or Hspa5), a master regulator of intracellular proteostasis, in two mouse models of neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD). To this end, we used mice bearing partial genetic deletion of the BiP gene (BiP^+/− mice), which, for the ALS model, were crossed with mutant SOD1 (mSOD1) transgenic mice to generate mSOD1/BiP^+/− double mutant mice. Our data revealed a more intense neurological decline in the double mutants, reflected in a greater deterioration of the neurological score and rotarod performance, with also a reduced animal survival, compared to mSOD1 transgenic mice. Such worsening was associated with higher microglial (labelled with Iba-1 immunostaining) and, to a lesser extent, astroglial (labelled with GFAP immunostaining) immunoreactivities found in the double mutants, but not with a higher loss of spinal motor neurons (labelled with Nissl staining) in the spinal cord. The morphological analysis of Iba-1 and GFAP-positive cells revealed a higher presence of activated cells, characterized by elevated cell body size and shorter processes, in double mutants compared to mSOD1 mice with normal BiP expression. In the case of the PD model, BiP^+/− mice were unilaterally lesioned with the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). In this case, however, we did not detect a greater susceptibility to damage in mutant mice, as the motor defects caused by 6-OHDA in the pole test and the cylinder rearing test, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity (labelled with CD68 and GFAP immunostaining) detected in the substantia nigra were of similar magnitude in BiP^+/− mice compared with wildtype animals. Therefore, our findings support the view that a dysregulation of the protein BiP may contribute to ALS pathogenesis. As BiP has been recently related to cannabinoid type-1 (CB₁) receptor function, our work also opens the door to future studies on a possible link between BiP and the neuroprotective effects of cannabinoids that have been widely reported in this neuropathological context. In support of this possibility, preliminary data indicate that CB₁ receptor levels are significantly reduced in mSOD1 mice having partial deletion of BiP gene.