Transgenic Mice Overexpressing Human STIM2 and ORAI1 in Neurons Exhibit Changes in Behavior and Calcium Homeostasis but Show No Signs of Neurodegeneration.

The maintenance of proper cytosolic Ca2+ level is crucial for neuronal survival, and dysregulation of Ca2+ homeostasis is found in a variety of neurological disorders, including Alzheimer's disease. According to the "Ca2+ hypothesis of aging", Ca2+ disturbances precede the onset of AD symptoms and lead to neurodegeneration. STIM and ORAI proteins are involved in neuronal physiological and pathological processes as essential components of the store-operated Ca2+ entry. Our previous data suggested that overexpression of STIM2 and ORAI1 might increase basal neuronal cytosolic Ca2+ level. We generated double transgenic mice overexpressing these two genes in neurons, expecting that the increased basal Ca2+ concentration will lead to premature neurodegeneration. We observed changes in Ca2+ homeostasis and electrophysiological properties in acute brain slices of STIM2/ORAI1 neurons. However, we did not observe any augmentation of neurodegenerative processes, as tested by Fluoro-Jade® C staining and assessment of amyloidogenesis. The battery of behavioral tests did not show any signs of accelerated aging. We conclude that changes of calcium homeostasis induced by overexpression of STIM2 and ORAI1 had no substantial adverse effects on neurons and did not lead to early neurodegeneration.

Changes in Calcium Homeostasis and Gene Expression Implicated in Epilepsy in Hippocampi of Mice Overexpressing ORAI1.

Previously, we showed that the overexpression of ORAI1 calcium channel in neurons of murine brain led to spontaneous occurrence of seizure-like events in aged animals of transgenic line FVB/NJ-Tg(ORAI1)Ibd (Nencki Institute of Experimental Biology). We aimed to identify the mechanism that is responsible for this phenomenon. Using a modified Ca2+-addback assay in the CA1 region of acute hippocampal slices and FURA-2 acetomethyl ester (AM) Ca2+ indicator, we found that overexpression of ORAI1 in neurons led to altered Ca2+ response. Next, by RNA sequencing (RNAseq) we identified a set of genes, whose expression was changed in our transgenic animals. These data were validated using customized real-time PCR assays and digital droplet PCR (ddPCR) ddPCR. Using real-time PCR, up-regulation of hairy and enhancer of split-5 (Hes-5) gene and down-regulation of aristaless related homeobox (Arx), doublecortin-like kinase 1 (Dclk1), and cyclin-dependent kinase-like 5 (Cdkl5, also known as serine/threonine kinase 9 (Stk9)) genes were found. Digital droplet PCR (ddPCR) analysis revealed down-regulation of Arx. In humans, ARX, DCLK1, and CDLK5 were shown to be mutated in some rare epilepsy-associated disorders. We conclude that the occurrence of seizure-like events in aged mice overexpressing ORAI1 might be due to the down-regulation of Arx, and possibly of Cdkl5 and Dclk1 genes.

Overexpression of STIM1 in neurons in mouse brain improves contextual learning and impairs long-term depression.

STIM1 is an endoplasmic reticulum calcium sensor that is involved in several processes in neurons, including store-operated calcium entry. STIM1 also inhibits voltage-gated calcium channels, such as Cav1.2 and Cav3.1, and is thus considered a multifunctional protein. The aim of this work was to investigate the ways in which transgenic neuronal overexpression of STIM1 in FVB/NJ mice affects animal behavior and the electrophysiological properties of neurons in acute hippocampal slices. We overexpressed STIM1 from the Thy1.2 promoter and verified neuronal expression by quantitative reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry. Mature primary hippocampal cultures expressed STIM1 but exhibited no changes in calcium homeostasis. Basal synaptic transmission efficiency and short-term plasticity were comparable in slices that were isolated from transgenic mice, similarly as the magnitude of long-term potentiation. However, long-term depression that was induced by the glutamate receptor 1/5 agonist (S)-3,5-dihydroxyphenylglycine was impaired in STIM1 slices. Interestingly, transgenic mice exhibited a decrease in anxiety-like behavior and improvements in contextual learning. In summary, our data indicate that STIM1 overexpression in neurons in the brain perturbs metabotropic glutamate receptor signaling, leading to impairments in long-term depression and alterations in animal behavior. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Interaction of myosin VI and its binding partner DOCK7 plays an important role in NGF-stimulated protrusion formation in PC12 cells.

DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1 GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski et al. (2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in other cell lines and demonstrated that MVI cargo domain via its RRL motif binds to DOCK7 C-terminal M2 and DHR2 domains. In MVI knockdown cells, lower Rac1 activity and a decrease of DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7 activity. MVI and DOCK7 co-localization was maintained during NGF-stimulated PC12 cell differentiation and observed also in the outgrowths. Also, during differentiation an increase in phosphorylation of DOCK7 as well as of its downstream effector JNK kinase was detected. Interestingly, overexpression of GFP-tagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that full length protein is important for this process. Moreover, a transient increase in Rac activity observed at 5min of NGF-stimulated differentiation of PC12 cells (overexpressing either GFP or GFP-MVI) was not detected in cells overexpressing the cargo domain. These data indicate that MVI-DOCK7 interaction could have functional implications in the protrusion outgrowth, and full length MVI seems to be important for delivery and maintenance of DOCK7 along the protrusions, and exerting its GEF activity.

SOCE in neurons: Signaling or just refilling?

In this review we describe the present knowledge about store operated Ca²âº entry (SOCE) in neurons and the proteins involved in this process: STIM, as well as Orai and TRP channels. We address the issue of whether SOCE is used only to refill Ca²âº in the ER or whether Ca²âº that enters the neuronal cell during SOCE also performs signaling functions. We collected the data indicating that SOCE and its components participate in the important processes in neurons. This has implications for identifying new drug targets for the treatment of brain diseases. Evidence indicates that in neurodegenerative diseases Ca²âº homeostasis and SOCE components become dysregulated. Thus, different targets and strategies might be identified for the potential treatment of these diseases. This article is part of a Special Issue entitled: 13th European symposium on calcium.

Involvement of unconventional myosin VI in myoblast function and myotube formation.

The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873-885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.

Myosin VI in skeletal muscle: its localization in the sarcoplasmic reticulum, neuromuscular junction and muscle nuclei.

Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309-314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 µm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.

Study on the Biodegradation of Poly(Butylene Succinate)/Wheat Bran Biocomposites.

This paper presents the results of a study investigating the biodegradation of poly(butylene succinate) (PBS)/wheat bran (WB) biocomposites. Injection mouldings were subjected to biodegradation in compost-filled bioreactors under controlled humidity and temperature conditions. The effects of composting time (14, 42 and 70 days) and WB mass content (10%, 30% and 50% wt.) on the structural and thermal properties of the samples were investigated. Measurements were made by infrared spectral analysis, scanning electron microscopy, differential scanning calorimetry, thermogravimetric analysis, and gel permeation chromatography. Results demonstrated that both the thermal and structural properties of the samples depended greatly on the biodegradation time. Specifically, their crystallinity degree increased significantly while molecular mass sharply decreased with biodegradation time, whereas their thermal resistance only showed a slight increase. This resulted from enzymatic hydrolysis that led to the breakdown of ester bonds in polymer chains. It was also found that a higher WB content led to a higher mass loss in the biocomposite samples during biodegradation and affected their post-biodegradation properties. A higher bran content increased the degree of crystallinity of the biocomposite samples but reduced their thermal resistance and molecular mass.

Lack of Stim2 Affects Vision-Dependent Behavior and Sensitivity to Hypoxia.

Stromal interaction molecules (STIMs) are endoplasmic reticulum-resident proteins that regulate Ca2+ homeostasis and signaling by store-operated calcium entry (SOCE). The different properties and functions of STIM1 and STIM2 have been described mostly based on work in vitro. STIM2 knockout mice do not survive until adulthood. Therefore, we generated and characterized stim2a and stim2b double-knockout zebrafish. The (stim2a;stim2b)-/- zebrafish did not have any apparent morphological phenotype. However, RNA sequencing revealed 1424 differentially expressed genes. One of the most upregulated genes was annexin A3a, which is a marker of activated microglia. This corresponded well to an increase in Neutral Red staining in the in vivo imaging of the (stim2a;stim2b)-/- zebrafish brain. The lack of Stim2 decreased zebrafish survival under low oxygen conditions. Behavioral tests, such as the visual-motor response test and dark-light preference test, indicated that (stim2a;stim2b)-/- larvae might have problems with vision. This was consistent with the downregulation of many genes that are related to light perception. The periodic acid-Schiff staining of retina sections from adult zebrafish revealed alterations of the stratum pigmentosum, suggesting the involvement of a Stim2-dependent process in visual perception. Altogether, these data reveal new functions for Stim2 in the nervous system.

Dock7: a GEF for Rho-family GTPases and a novel myosin VI-binding partner in neuronal PC12 cells.

Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI-Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization.

Flammability of Polymer Compositions Filled with Wheat Bran.

The article presents the results of flammability tests on polymer compositions with wheat bran (WB) as the applied filler, and polyethylene (PE) or poly(butylene succinate) (PBS) as the matrix material. Tests were conducted using samples of compositions containing 10, 30 and 50%wt wheat bran. The test samples were manufactured by injection moulding from compositions previously produced by extrusion pelleting. For comparative purposes, samples made only of the plastics used for the composition matrix were also examined. Flammability tests were carried out in accordance with the recommendations of EN 60695-11-10 Part 11-10 with horizontal and vertical positioning of the sample, using a universal flammability-test-stand. During the flammability tests, changes in the temperature field in the area of the burning sample were also recorded, using a thermal imaging camera. Sample residues after flammability tests were also examined with infrared spectroscopy (FTIR) to assess their thermal destruction. The results of the study showed a significant increase in flammability with bran content for both PE and PBS matrix compositions. Clear differences were also found in the combustion behaviour of the matrix materials alone. Both the burning rate and maximum flame temperature were lower in favour of PBS. PBS compositions with wheat bran also showed lower flammability, compared with their PE matrix counterparts.

Loss of Unconventional Myosin VI Affects cAMP/PKA Signaling in Hindlimb Skeletal Muscle in an Age-Dependent Manner.

Myosin VI (MVI) is a unique unconventional myosin ubiquitously expressed in metazoans. Its diverse cellular functions are mediated by interactions with a number of binding partners present in multi-protein complexes. MVI is proposed to play important roles in muscle function and myogenesis. Previously, we showed that MVI is present in striated muscles and myogenic cells, and MVI interacts with A-kinase anchoring protein 9 (AKAP9), a scaffold for PKA and its regulatory proteins. Since PKA directly phosphorylates the MVI cargo binding domain, we hypothesized that the cellular effects of MVI are mediated by the cAMP/PKA signaling pathway, known to play important roles in skeletal muscle metabolism and myogenesis. To elucidate the potential role of MVI in PKA signaling in hindlimb muscle function, we used mice lacking MVI (Snell's waltzer, SV), considered as natural MVI knockouts, and heterozygous littermates. We used muscles isolated from newborn (P0) as well as 3- and 12-month-old adult mice. We observed a significant increase in the muscle to body mass ratio, which was most evident for the soleus muscle, as well as changes in fiber size, indicating alterations in muscle metabolism. These observations were accompanied by age-dependent changes in the activity of PKA and cAMP/PKA-dependent transcriptional factor (CREB). Additionally, the levels of adenylate cyclase isoforms and phosphodiesterase (PDE4) were age-dependent. Also, cAMP levels were decreased in the muscle of P0 mice. Together, these observations indicate that lack of MVI impairs PKA signaling and results in the observed alterations in the SV muscle metabolism, in particular in newborn mice.

Myosin VI in PC12 cells plays important roles in cell migration and proliferation but not in catecholamine secretion.

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release.

Modern Biodegradable Plastics-Processing and Properties Part II.

Four different plastics were tested: potato starch based plastic (TPS-P)-BIOPLAST GF 106/02; corn starch based plastic (TPS-C)-BioComp BF 01HP; polylactic acid (polylactide) plastic (PLA)-BioComp BF 7210 and low density polyethylene, trade name Malen E FABS 23-D022; as a petrochemical reference sample. Using the blown film extrusion method and various screw rotational speeds, films were obtained and tested, as a result of which the following were determined: breaking stress, strain at break, static and dynamic friction coefficient of film in longitudinal and transverse direction, puncture resistance and strain at break, color, brightness and gloss of film, surface roughness, barrier properties and microstructure. The biodegradable plastics tested are characterized by comparable or even better mechanical strength than petrochemical polyethylene for the range of film blowing processing parameters used here. The effect of the screw rotational speed on the mechanical characteristics of the films obtained was also demonstrated. With the increase in the screw rotational speed, the decrease of barrier properties was also observed. No correlation between roughness and permeability of gases and water vapor was shown. It was indicated that biodegradable plastics might be competitive for conventional petrochemical materials used in film blowing niche applications where cost, recyclability, optical and water vapor barrier properties are not critical.

Experimental Determination of Coefficients for the Renner Model of the Thermodynamic Equation of State of the Poly(butylene succinate) and Wheat Bran Biocomposites.

This paper presents the assumptions of a thermodynamic equation of state for polymers according to the Renner model. The experiments involved extruding a biocomposite based on poly(butylene succinate) that was filled with ground wheat bran with its size not exceeding 200 µm. The biocomposite was produced in pellet form with three different contents by weight of wheat bran, i.e., 10%, 30% and 50%. All specimens were examined for their thermodynamic p-v-T characteristics. Taking advantage of the SimFit module of Cadmould 3D-F, experimental results were used to determine the coefficients of thermodynamic equation of state for the tested biocomposite according to the Renner model. The coefficients were then used to calculate transition temperature and to create diagrams illustrating the relationship between pressure, temperature and specific volume for the tested biocomposite. The obtained results can serve as a basis for assessing the suitability of the biocomposite for injection molding, selecting technological parameters of this process, as well as for analyzing shrinkage and defects of injection-molded parts.

Annexin A3: a newly identified player in store­operated calcium entry.

Store­operated calcium entry (SOCE) is important for refilling endoplasmic reticulum (ER) Ca2+ stores and Ca2+ signaling. Extracellular Ca2+ is conducted by highly Ca2+­selective Orai channels that are activated by stromal interaction molecule proteins (STIMs), which are sensitive ER Ca2+ sensors. We found an approximately five­fold increase in annexin A3a (anxa3a) expression levels in stim2b knockout zebrafish. The present study investigated whether annexin A3 protein is involved in SOCE. We used Ca2+ imaging and electrophysiological recordings to determine the effect of annexin A2, A3, and A6 overexpression on SOCE and Orai-dependent Ca2+ currents (ICRAC) in cultured cells. Annexin A3 increased SOCE amplitude and potentiated Ca2+ ICRAC currents. These results suggest that annexin A3 is a positive modulator of SOCE.

Analysis of Selected Properties of Injection Moulded Sustainable Biocomposites from Poly(butylene succinate) and Wheat Bran.

The paper presents a procedure of the manufacturing and complex analysis of the properties of injection mouldings made of polymeric composites based on the poly(butylene succinate) (PBS) matrix with the addition of a natural filler in the form of wheat bran (WB). The scope of the research included measurements of processing shrinkage and density, analysis of the chemical structure, measurements of the thermal and thermo-mechanical properties (Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TG), Heat Deflection Temperature (HDT), and Vicat Softening Temperature (VST)), and measurements of the mechanical properties (hardness, impact strength, and static tensile test). The measurements were performed using design of experiment (DOE) methods, which made it possible to determine the investigated relationships in the form of polynomials and response surfaces. The mass content of the filler and the extruder screw speed during the production of the biocomposite granulate, which was used for the injection moulding of the test samples, constituted the variable factors adopted in the DOE. The study showed significant differences in the processing, thermal, and mechanical properties studied for individual systems of the DOE.

Artificial Ageing, Chemical Resistance, and Biodegradation of Biocomposites from Poly(Butylene Succinate) and Wheat Bran.

The results of comprehensive studies on accelerated (artificial) ageing and biodegradation of polymer biocomposites on PBS matrix filled with raw wheat bran (WB) are presented in this paper. These polymer biocomposites are intended for the manufacture of goods, in particular disposable packaging and disposable utensils, which decompose naturally under the influence of biological agents. The effects of wheat bran content within the range of 10-50 wt.% and extruder screw speed of 50-200 min-1 during the production of biocomposite pellets on the resistance of the products to physical, chemical, and biological factors were evaluated. The research included the determination of the effect of artificial ageing on the changes of structural and thermal properties by infrared spectra (FTIR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TG). They showed structural changes-disruption of chains within the ester bond, which occurred in the composition with 50% bran content as early as after 250 h of accelerated ageing. An increase in the degree of crystallinity with ageing was also found to be as high as 48% in the composition with 10% bran content. The temperature taken at the beginning of weight loss of the compositions studied was also lowered, even by 30 °C at the highest bran content. The changes of mechanical properties of biocomposite samples were also investigated. These include: hardness, surface roughness, transverse shrinkage, weight loss, and optical properties: colour and gloss. The ageing hardness of the biocomposite increased by up to 12%, and the surface roughness (Ra) increased by as much as 2.4 µm at the highest bran content. It was also found that ageing causes significant colour changes of the biocomposition (ΔE = 7.8 already at 10% bran content), and that the ageing-induced weight loss of the biocomposition of 0.31-0.59% is lower than that of the samples produced from PBS alone (1.06%). On the other hand, the transverse shrinkage of moldings as a result of ageing turned out to be relatively small, at 0.05%-0.35%. The chemical resistance of biocomposites to NaOH and HCl as well as absorption of polar and non-polar liquids (oil and water) were also determined. Biodegradation studies were carried out under controlled conditions in compost and weight loss of the tested compositions was determined. The weight of samples made from PBS alone after 70 days of composting decreased only by 4.5%, while the biocomposition with 10% bran content decreased by 15.1%, and with 50% bran, by as much as 68.3%. The measurements carried out showed a significant influence of the content of the applied lignocellulosic fillers (LCF) in the form of raw wheat bran (WB) on the examined properties of the biocompositions and the course of their artificial ageing and biodegradation. Within the range under study, the screw speed of the extruder during the production of biocomposite pellets did not show any significant influence on most of the studied properties of the injection mouldings produced from it.

Efficiency of Twin-Screw Extrusion of Biodegradable Poly (Butylene Succinate)-Wheat Bran Blend.

Unmodified poly (butylene succinate) (PBS) is characterized by very good processability; however, after the incorporation of various fillers of plant origin, its processing becomes much more complicated and its properties are significantly affected. Detailed studies of the processing aspects of PBS/wheat bran (WB) biocomposition are lacking, despite the addition of WB having a significant impact on both the production efficiency and the properties of end products. This research paper presents test results of the co-rotating twin-screw extrusion processing of a biodegradable polymer blend, the matrix of which was PBS, with WB as the filler. In undertaking this task, we examined the impact of extruder screw rotational speed and WB content on the characteristics of extrusion processing, as well as on certain thermal, physical, structural and processing properties of the obtained blend. The WB introduced to the blend was in the form of a selected fraction with particles smaller than 0.2 mm. The measurements were conducted using the Design of Experiment (DOE) methods, which enabled establishing the studied relationships in the form of polynomials and response surfaces. The determined extrusion process characteristics covered the impact of screw rotational speed and WB content on the mass flow rate of the processed blend and its pressure, the screw drive torque and specific energy consumption. The studies of the obtained polymer blend included determining the impact of the aforementioned variable factors on the melt flow rate (MFR) index, chemical structure (FTIR), thermal properties (differential scanning calorimetry (DSC), thermogravimetry (TG), derivative thermogravimetry (DTG)), p-v-T relationships, microstructure, density and moisture absorbance. Analysis of variance (ANOVA) was used to assess the effect of individual variable factors. The results of this work are presented, inter alia, using Pareto charts of standardized effects, which illustrate the influence of individual terms of the determined regression equations on the studied quantity.

npc2-Deficient Zebrafish Reproduce Neurological and Inflammatory Symptoms of Niemann-Pick Type C Disease.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disease that is caused by a mutation of the NPC1 or NPC2 gene, in which un-esterified cholesterol and sphingolipids accumulate mainly in the liver, spleen, and brain. Abnormal lysosomal storage leads to cell damage, neurological problems, and premature death. The time of onset and severity of symptoms of NPC disease are highly variable. The molecular mechanisms that are responsible for NPC disease pathology are far from being understood. The present study generated and characterized a zebrafish mutant that lacks Npc2 protein that may be useful for studies at the organismal, cellular, and molecular levels and both small-scale and high-throughput screens. Using CRISPR/Cas9 technology, we knocked out the zebrafish homolog of NPC2. Five-day-old npc2 mutants were morphologically indistinguishable from wildtype larvae. We found that live npc2-/- larvae exhibited stronger Nile blue staining. The npc2-/- larvae exhibited low mobility and a high anxiety-related response. These behavioral changes correlated with downregulation of the mcu (mitochondrial calcium uniporter) gene, ppp3ca (calcineurin) gene, and genes that are involved in myelination (mbp and mpz). Histological analysis of adult npc2-/- zebrafish revealed that pathological changes in the nervous system, kidney, liver, and pancreas correlated with inflammatory responses (i.e., the upregulation of il1, nfκß, and mpeg; i.e., hallmarks of NPC disease). These findings suggest that the npc2 mutant zebrafish may be a model of NPC disease.