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Hyperhaemolysis syndrome, often associated with sickle cell disease, can also occur in non sickle cell conditions following viral infections and autoimmune disorders. This case report presents a rare instance of hyperhaemolysis in the context of familial anaemia. A 41-year-old female with a history of unexplained anaemia presented with four episodes of haematemesis, abdominal pain, and headache. She had received a blood transfusion a month prior. Upon admission, she was diagnosed with pancytopenia and hypovolemic shock and was treated with packed cell volume transfusions. The patient had a significant family history of anaemia in her sisters, who also required intermittent blood transfusions. Following admission, she developed thrombocytopenia, a drop in Haemoglobin (Hb), petechiae, and subdural haemorrhage. Further investigations confirmed hyperhaemolysis syndrome. Treatment included blood transfusions, random donor platelet transfusions, and corticosteroids. The patient was discharged on prednisolone and cyclosporine. Over six months, her condition improved significantly, with no further bleeding episodes and stable Hb levels. Diagnosing hyperhaemolysis syndrome is challenging and requires a high index of suspicion. This case underscores the importance of considering hyperhaemolysis in patients with familial anaemia and unexplained haemolytic episodes. Early recognition and appropriate management are crucial for favourable outcomes.

Hyperhaemolysis syndrome, often associated with sickle cell disease, can also occur in non sickle cell conditions following viral infections and autoimmune disorders. This case report presents a rare instance of hyperhaemolysis in the context of familial anaemia. A 41-year-old female with a history of unexplained anaemia presented with four episodes of haematemesis, abdominal pain, and headache. She had received a blood transfusion a month prior. Upon admission, she was diagnosed with pancytopenia and hypovolemic shock and was treated with packed cell volume transfusions. The patient had a significant family history of anaemia in her sisters, who also required intermittent blood transfusions. Following admission, she developed thrombocytopenia, a drop in Haemoglobin (Hb), petechiae, and subdural haemorrhage. Further investigations confirmed hyperhaemolysis syndrome. Treatment included blood transfusions, random donor platelet transfusions, and corticosteroids. The patient was discharged on prednisolone and cyclosporine. Over six months, her condition improved significantly, with no further bleeding episodes and stable Hb levels. Diagnosing hyperhaemolysis syndrome is challenging and requires a high index of suspicion. This case underscores the importance of considering hyperhaemolysis in patients with familial anaemia and unexplained haemolytic episodes. Early recognition and appropriate management are crucial for favourable outcomes. [ABSTRACT FROM AUTHOR]

Methylcarbamate (MC), a reaction product between dimethyl dicarbonate and ammonia or ammonium ion, is a potent hepatocarcinogen in F344 rats. Various genotoxicity tests have shown negative results for MC. Although previous studies have described the effects of MC on the liver, including the formation of characteristic basophilic cytoplasmic inclusions (CIs) in hepatocytes, the toxicological significance of CIs and their involvement in hepatocarcinogenesis remain unclear. In the current study, to elucidate the mechanisms of MC hepatocarcinogenesis, we examined hepatotoxicity and genotoxicity after 4 weeks of administration of MC using gpt delta rats with an F344 genetic background as a reporter gene transgenic animal model. Histopathologically, single-cell necrosis, karyomegaly, and the formation of CIs positive for Feulgen staining were observed in hepatocytes at the carcinogenic dose, demonstrating the hepatotoxicity of MC. CIs were also detected as large micronuclei in liver micronucleus tests but not in the bone marrow, suggesting that MC could cause chromosomal instability specifically in the livers of rats. Reporter gene mutation assays demonstrated that MC did not induce mutagenicity even in the liver. Immunofluorescence analyses revealed that CIs exhibited loss of nuclear envelope integrity, increased heterochromatinization, and accumulation of DNA damage. An increase in liver STING protein levels suggested an effect on the cyclic GMP-AMP synthase/stimulator of interferon genes innate immune pathway. Overall, these data demonstrated the possible occurrence of chromothripsis-like chromosomal rearrangements via CIs. Thus, the formation of CIs could be a crucial event in the early stage of MC-induced hepatocarcinogenesis in F344 rats. [ABSTRACT FROM AUTHOR]

Background: The rapid development of automatic blood cell analyzers has greatly optimized complete blood count results. However, erroneous results relevant to automatic blood cell analyzers still exist. Pseudothrombocytopenia can be observed in both cases of anticoagulant-induced platelet aggregation, and the presence of large and giant platelets. Methods: A rare case of a MYH9-related disorder, in which marked underestimation of platelet count was led by large and giant platelets using the impedance count by an automated hematology analyzer. Moreover, lancetshaped and Dohle body-like cytoplasmic inclusions were detected in almost all white blood cells of the patient. Results: The platelet count was done by an optical platelet counter or a fluorescence platelet counter, and peripheral blood smear was evaluated. In addition, the diagnosis of MYH9-related disorder was established by the molecular findings. Conclusions: Identification of the peripheral blood smear and familial history will eliminate the need for further laboratory testing and bone marrow examination. [ABSTRACT FROM AUTHOR]

Mutations in Fused-in-Sarcoma (FUS) gene involving the nuclear localization signal (NLS) domain lead to juvenile-onset Amyotrophic Lateral Sclerosis (ALS). The mutant protein mislocalizes to the cytoplasm, incorporating it into Stress Granules (SG). Whether SGs are the first step to the formation of stable FUS-containing aggregates is still unclear. In this work, we used acute and chronic stress paradigms to study the SG dynamics in a human SH-SY5Y neuroblastoma cell line carrying a deletion of the NLS domain of the FUS protein (homozygous: ΔNLS–/–; heterozygous: ΔNLS+/–). Wild-type (WT) cells served as controls. We evaluated the subcellular localization of the mutant protein through immunoblot and immunofluorescence, in basal conditions and after acute stress and chronic stress with sodium arsenite (NaAsO2). Cells were monitored for up to 24 h after rescue. FUS was expressed in both nucleus and cytoplasm in the ΔNLS+/– cells, whereas it was primarily cytoplasmic in the ΔNLS–/–. Acute NaAsO2 exposure induced SGs: at rescue,>90% of ΔNLS cells showed abundant FUS-containing if compared to less than 5% of the WT cells. The proportion of FUS-positive SGs remained 15–20% at 24 h in mutant cells. Cycloheximide did not abolish the long-lasting SGs in mutant cells. Chronic exposure to NaAsO2 did not induce significant SGs formation. A wealth of research has demonstrated that ALS-associated FUS mutations at the C-terminus facilitate the incorporation of the mutant protein into SGs. We have shown here that mutant FUS-containing SGs tend to fail to dissolve after stress, facilitating a liquid-to-solid phase transition. The FUS-containing inclusions seen in the dying motor neurons might therefore directly derive from SGs. This might represent an attractive target for future innovative therapies.

Mutations in Fused-in-Sarcoma (FUS) gene involving the nuclear localization signal (NLS) domain lead to juvenile-onset Amyotrophic Lateral Sclerosis (ALS). The mutant protein mislocalizes to the cytoplasm, incorporating it into Stress Granules (SG). Whether SGs are the first step to the formation of stable FUS-containing aggregates is still unclear. In this work, we used acute and chronic stress paradigms to study the SG dynamics in a human SH-SY5Y neuroblastoma cell line carrying a deletion of the NLS domain of the FUS protein (homozygous: ΔNLS–/–; heterozygous: ΔNLS+/–). Wild-type (WT) cells served as controls. We evaluated the subcellular localization of the mutant protein through immunoblot and immunofluorescence, in basal conditions and after acute stress and chronic stress with sodium arsenite (NaAsO2). Cells were monitored for up to 24 h after rescue. FUS was expressed in both nucleus and cytoplasm in the ΔNLS+/– cells, whereas it was primarily cytoplasmic in the ΔNLS–/–. Acute NaAsO2 exposure induced SGs: at rescue,>90% of ΔNLS cells showed abundant FUS-containing if compared to less than 5% of the WT cells. The proportion of FUS-positive SGs remained 15–20% at 24 h in mutant cells. Cycloheximide did not abolish the long-lasting SGs in mutant cells. Chronic exposure to NaAsO2 did not induce significant SGs formation. A wealth of research has demonstrated that ALS-associated FUS mutations at the C-terminus facilitate the incorporation of the mutant protein into SGs. We have shown here that mutant FUS-containing SGs tend to fail to dissolve after stress, facilitating a liquid-to-solid phase transition. The FUS-containing inclusions seen in the dying motor neurons might therefore directly derive from SGs. This might represent an attractive target for future innovative therapies. [ABSTRACT FROM AUTHOR]

Amyotrophic Lateral Sclerosis (ALS) is characterized by degeneration of motor neurons in the brain and spinal cord. Cytoplasmic inclusions of TDP-43 are frequently reported in motor neurons of ALS patients. TDP-43 has also been shown to associate with stress granules (SGs), a complex of proteins and mRNAs formed in response to stress stimuli that temporarily sequester mRNA translation. The effect of pathogenic TDP-43 mutations within glycine-rich regions (where the majority of ALS-causing TDP-43 mutations occur) on SG dynamics in motor neurons is poorly understood. To address this issue, we generated murine NSC-34 cell lines that stably over-express wild type TDP-43 (TDP-43WT) or mutant forms (ALS-causing TDP-43 mutations TDP-43A315T or TDP-43M337V). We then differentiated these NSC-34 lines into motoneuron-like cells and evaluated SG formation and disassembly kinetics in response to oxidative or osmotic stress treatment. Wild type and mutant TDP-43 appeared to be largely retained in the nucleus following exposure to arsenite-induced oxidative stress. Upon arsenite removal, mutant TDP-43 clearly accumulated within HuR positive SGs in the cytoplasm, whereas TDP-43WT remained mostly within the nucleus. 24 h following arsenite removal, all SGs were disassembled in both wild type and mutant TDP-43 expressing cells. By contrast, we observed significant differences in the dynamics of mutant TDP-43 association with SGs in response to hyperosmotic stress. Specifically, in response to sorbitol treatment, TDP-43WT remained in the nucleus, whereas mutant TDP-43 relocalized to HuR positive SGs in the cytoplasm following exposure to sorbitol stress, resulting in a significant increase in TDP-43 SG numbers. These SGs remained assembled for 24 h following removal of sorbitol. Our data reveal that under certain stress conditions the rates of SG formation and disassembly is modulated by TDP-43 mutations associated with ALS, and suggest that this may be an early event in the seeding of insoluble cytoplasmic inclusions observed in ALS.

Background and Objectives: This study aims to develop and evaluate NeuNN, a system based on convolutional neural networks (CNN) and generative adversarial networks (GAN) for the automatic identification of normal neutrophils and those containing several types of inclusions or showing hypogranulation., Methods: From peripheral blood smears, a set of 5605 digital images was obtained with neutrophils belonging to seven categories: Normal neutrophils (NEU), Hypogranulated (HYP) or containing cryoglobulins (CRY), Döhle bodies (DB), Howell-Jolly body-like inclusions (HJBLI), Green-blue inclusions of death (GBI) and phagocytosed bacteria (BAC). The dataset utilized in this study has been made publicly available. The class of GBI was augmented using synthetic images generated by GAN. The NeuNN classification model is based on an EfficientNet-B7 architecture trained from scratch., Results: NeuNN achieved an overall performance of 94.3% accuracy on the test data set. Performance metrics, including sensitivity, specificity, precision, F1-Score, Jaccard index, and Matthews correlation coefficient indicated overall values of 94%, 99.1%, 94.3%, 94.3%, 89.6%, and 93.6%, respectively., Conclusions: The proposed approach, combining data augmentation and classification techniques, allows for automated identification of morphological findings in neutrophils, such us inclusions or hypogranulation. The system can be used as a support tool for clinical pathologists to detect these specific abnormalities with clinical relevance., Competing Interests: Declaration of competing interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

IntroductionMutations in the valosin-containing protein (VCP) gene cause hereditary inclusion body myopathy (IBM) associated with Paget disease of bone (PDB), and frontotemporal dementia (FTD). More recently, these mutations have been linked to 2% of familial amyotrophic lateral sclerosis (ALS) cases. A knock-in mouse model offers the opportunity to study VCP-associated pathogenesis.MethodsThe VCP(R155H/+) knock-in mouse model was assessed for muscle strength and immunohistochemical, Western blot, apoptosis, autophagy, and microPET/CT imaging analyses.ResultsVCP(R155H/+) mice developed significant progressive muscle weakness, and the quadriceps and brain developed progressive cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies, and increased LC3-II staining. MicroCT analyses revealed Paget-like lesions at the ends of long bones. Spinal cord demonstrated neurodegenerative changes, ubiquitin, and TDP-43 pathology of motor neurons.ConclusionsVCP(R155H/+) knock-in mice represent an excellent preclinical model for understanding VCP-associated disease mechanisms and future treatments.

The resistance of microorganisms to heavy metals in polluted environments is mediated by genetically determined mechanisms. One such mechanism includes the intracellular sequestration of heavy metals in polyphosphate (polyP) inclusions. In Cr(III) contaminated mediums, Ochrobactrum anthropi DE2010 is able to bind and sequester Cr(III) in polyP inclusions. In order to further study the relationship between Cr(III) tolerance and polyP production in O. anthropi DE2010, we carried out whole genomic sequencing, analysis of single nucleotide polymorphisms (SNPs), polyP chemical quantification, and determination of the relative abundance and morphometry of polyP inclusions. In the O. anthropi DE2010 genome, six polyP and pyrophosphate (PPi) metabolic genes were found. Furthermore, genomic analysis via SNPs calling revealed that O. anthropi ATCC49188 and DE2010 strains had average variations of 1.51% in their whole genome sequences and 1.35% variation associated with the principal polyP metabolic gene cluster. In addition, the accumulation of polyP in the DE2010 strain and number of polyP inclusions found were directly correlated with the concentration of Cr(III) in contaminated cultures. The results presented in this study may enhance the understanding of polyP production in response to Cr(III) toxicity in the O. anthropi DE2010 strain. This knowledge may facilitate the successful removal of Cr(III) from the natural environment. [ABSTRACT FROM AUTHOR]

Abstract It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease.

Clinical History: A 57‐year old woman presented with left hand pain, periodic leg movement during sleep, gradual onset of stiffness, clumsiness, and falls. Neurological examination showed: generalized rigidity and bradykinesia. There was left hand dystonic posturing and ideomotor apraxia, as well as mirror movements of upper limbs and stimulus‐sensitive myoclonus. The patient had a high‐pitched voice and hypophonia (Video S1). Discussion: Experts discuss localization and the syndromic diagnosis and predict the underlying pathology. The pathological diagnosis is then provided and clinical learning points are considered. View Supplementary Video 1 [ABSTRACT FROM AUTHOR]

We have previously shown that a single application of the growth factors ciliary neurotrophic factor (CNTF) or fibroblast growth factor 2 (FGF-2) to the crushed optic nerve of the frog, Rana pipiens, increases the numbers and elongation rate of regenerating retinal ganglion cell axons. Here we investigate the effects of these factors on the numbers and types of macrophages that invade the regeneration zone. In control PBS-treated nerves, many macrophages are present 100 μm distal to the crush site at 1 week after injury; their numbers halve by 2 weeks. A single application of CNTF at the time of injury triples the numbers of macrophages at 1 week, with this increase compared to control being maintained at 2 weeks. Application of FGF-2 is equally effective at 1 week, but the macrophage numbers have fallen to control levels at 2 weeks. Immunostaining with a pan-macrophage marker, ED1, and a marker for M2-like macrophages, Arg-1, showed that the proportion of the putative M2 phenotype remained at approximately 80% with all treatments. Electron microscopy of the macrophages at 1 week shows strong phagocytic activity with all treatments, with many vacuoles containing axon fragments and membrane debris. At 2 weeks with PBS or FGF-2 treatment the remaining macrophages are less phagocytically active, containing mainly lipid inclusions. With CNTF treatment, at 2 weeks many of the more numerous macrophages are still phagocytosing axonal debris, although they also contain lipid inclusions. We conclude that the increase in macrophage influx seen after growth factor application is beneficial for the regenerating axons, probably due to more extensive removal of degenerating distal axons, but also perhaps to secretion of growth-promoting substances. [ABSTRACT FROM AUTHOR]

Abstract Neoplastic B cells may have cytoplasmic inclusions that are visible in routine peripheral blood smears by light microscopy.

Autophagy plays a major role in the elimination of cellular waste components, the renewal of intracellular proteins and the prevention of the build-up of redundant or defective material. It is fundamental for the maintenance of homeostasis and especially important in post-mitotic neuronal cells, which, without competent autophagy, accumulate protein aggregates and degenerate. Many neurodegenerative diseases are associated with defective autophagy; however, whether altered protein turnover or accumulation of misfolded, aggregate-prone proteins is the primary insult in neurodegeneration has long been a matter of debate. Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by selective degeneration of motor neurons. Most of the ALS cases occur in sporadic forms (SALS), while 10%–15% of the cases have a positive familial history (FALS). The accumulation in the cell of misfolded/abnormal proteins is a hallmark of both SALS and FALS, and altered protein degradation due to autophagy dysregulation has been proposed to contribute to ALS pathogenesis. In this review, we focus on the main molecular features of autophagy to provide a framework for discussion of our recent findings about the role in disease pathogenesis of the ALS-linked form of the VAPB gene product, a mutant protein that drives the generation of unusual cytoplasmic inclusions.

Immune defense is a key feature in the life history of organisms, expensive to maintain, highly regulated by individuals and exposed to physiological and evolutionary trade-offs. In chelicerates, relatively scarce are the studies that relate postcopulatory mechanisms and immune response parameters. This work makes an approximation to the female’s immunological consequences produced after the placement of a foreign body in the genitalia of three scorpions species, two species that normally receive genital plugs during mating (Urophonius brachycentrus and U. achalensis) and one that does not (Zabius fuscus). Here we performed the first morphological description of the natural plugs of the two Urophonius species. We described complex three zoned structure anchored to the female genital atrium and based on this information we placed implants in the genitalia (for eliciting the local immune response) of virgin females of the three species and measured the immune encapsulation response to this foreign body. We found a greater and heterogeneous response in different zones of the implants in the plug producing species. To corroborate the specificity of this immune response, we compared the local genital reaction with the triggered response at a systemic level by inserting implants into the female body cavity of U. brachycentrus and Zabius fuscus. We found that the systemic response did not differ between species and that only in the plug producing species the local response in the genitalia was higher than the systemic one. We also compared the total hemocyte load before and after the genital implantation to see if this parameter was compromised by the immunological challenge. We confirmed that in Urophonius species the presence of a strange body in the genitalia caused a decrease in the hemocyte load. Besides, we find correlations between the body weight and the immunological parameters, as well as between different immunological parameters with each other. Complementarily, we characterized the hemocytes of the three scorpion species for the first time. This comparative study can help to provide a wider framework of the immunological characteristics of the species, their differences and their relationship with the particular postcopulatory mechanism such as the genital plugs. [ABSTRACT FROM AUTHOR]

TDP-43, an RNA-binding protein that is primarily nuclear and important in splicing and RNA metabolism, is mislocalized from the nucleus to the cytoplasm of neural cells in amyotrophic lateral sclerosis (ALS), and contributes to disease. We sought to investigate whether TDP-43 is mislocalized in infections with the acute neuronal GDVII strain and the persistent demyelinating DA strain of Theiler’s virus murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus of Picornaviridae because: i) L protein of both strains is known to disrupt nucleocytoplasmic transport, including transport of polypyrimidine tract binding protein, an RNA-binding protein, ii) motor neurons and oligodendrocytes are targeted in both TMEV infection and ALS. TDP-43 phosphorylation, cleavage, and cytoplasmic mislocalization to an aggresome were observed in wild type TMEV-infected cultured cells, with predicted splicing abnormalities. In contrast, cells infected with DA and GDVII strains that have L deletion had rare TDP-43 mislocalization and no aggresome formation. TDP-43 mislocalization was also present in neural cells of TMEV acutely-infected mice. Of note, TDP-43 was mislocalized six weeks after DA infection to the cytoplasm of oligodendrocytes and other glial cells in demyelinating lesions of spinal white matter. A recent study showed that TDP-43 knock down in oligodendrocytes in mice led to demyelination and death of this neural cell [], suggesting that TMEV infection mislocalization of TDP-43 and other RNA-binding proteins is predicted to disrupt key cellular processes and to contribute to the pathogenesis of TMEV-induced diseases. Drugs that inhibit nuclear export may have a role in antiviral therapy. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the effect of feeding licuri cake to lambs on the sensory characteristics, physicochemical characteristics and fatty acid (FA) profile of meat from lambs. Forty-four crossbred Santa Ines lambs (21.2 ± 2.70 kg body weight; 6 months old) were housed in individual pens and fed 4 experimental diets, containing 0, 8, 16 or 24% licuri cake (DM basis). The averages concentrations of ash (11.4), pH (5.82), lightness (38.1), cooking loss (26.8) or shear-force resistance (2.48) of lamb meat were not affected by the licuri cake diets. However, there was a linear decrease (P < 0.01) of redness and chroma indexes, lipid and protein contents, whereas the moisture content of the meat (P < 0.001) increased linearly due to the inclusion of licuri cake in lambs’ diets. The licuri cake inclusion in the lambs feed linearly increased (P < 0.05) the fatty acids concentrations of C12:0, C17:0, C20:0, C20:1, C18:3, C20:3, C20:4 and ΣPUFA/ΣMUFA ratio, Σω–3 and atherogenicity index (AI). However, C18:1 cis, C20:2, C20:5, ΣMUFA, ΣMUFA/ΣSFA and Σω–6:Σω–3 ratios in the longissimus lumborum of lambs linearly decreased by licuri cake inclusion. There was a quadratic increase (P < 0.05) on C14:0 (maximum point 4.94 g/100 g FAME to 14.5% licuri inclusion), C16:1 (maximum point 8.59 g/100 g FAME to 10.7% licuri inclusion) and enzymatic activities of Δ9-desaturase C16 (maximum point 27.5 g/100 g FAME to 10.6% licuri inclusion) in the longissimus lumborum of lambs fed due to increased concentrations of licuri cake. However, there was a quadratic decrease (P = 0.04) in ΣPUFA/ΣSFA ratio with minimum concentration of 0.63 g/100 g FAME to 11.1% inclusion. The inclusion of licuri cake in the lambs diet did not change (P > 0.05) the concentrations of SFA C10:0, C15:0, C16:0, C18:0, C14:1, MUFA C18:1 trans, PUFA C18:2 cis, CLA, total sum of ΣSFA and ΣPUFA, desirable fatty acids (DFA), hypocholesterolemic:hypercholesterolemic index, and elongase and Δ9-desaturase C18 enzymes. Licuri cake in the lamb diet improved (P < 0.05) meat aroma, flavor and overall acceptance by consumers. Licuri cake inclusion in the diet of lambs improves sensory attributes of meat and the meat fatty acid profile becomes nutritionally healthier for the human diet because do not affect major FA of meat; however, the growth performance of finishing lambs is reduced. [ABSTRACT FROM AUTHOR]

Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate “healthy” control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington’s disease. [ABSTRACT FROM AUTHOR]

Pseudozyma flocculosa is a fungus very useful and highly efficient as a biocontrol agent against powdery mildew. The reproduction of this fungus occurs exclusively by asexual production of conidia or sporidia that are the most suitable form for agricultural use and seems to be the most resistant to storage conditions. Despite the advantages offered by P. flocculosa in biological control, the use of this fungus use remains largely limited compared to that of chemical fungicides, at least partly due to the difficulty to obtain sporidia resistant to adverse environmental stresses in submerged culture conditions. Under solid-state and submerged-state cultivation, P. flocculosa strain CBS 16788 produced different types of sporidia. The submerged sporidia (SS) appeared relatively uniform in size, which was 15,4 ± 1,6 μm μm long, and 2,8 ± 0.8 μm wide. The aerial sporidia (AS) varied in shape and size, with a mean length of 8,2 ± 3 μm and width of 2,3 ± 0.6 μm. Under scanning and transmission electron microscopy, the cell wall of submerged sporidia was thinner than that of aerial spores, and the surface was smooth in contrast to the aerial sporidia that had a tendency to have verrucous, brittle surface characteristics. The thickness of the aerial sporidia wall is due to the presence of an outer layer rich in melanin. The sporidia germination was compared on YMPD (yeast extract, malt extract, soy peptone, dextrose and agar) coated coverslips. The aerial sporidia did not show germ tubes until 5 h of incubation, while the submerged sporidia showed many germ tubes after the same time. The resistance against the adverse environmental conditions in relation to the type of sporidia of P. flocculosa is discussed. [ABSTRACT FROM AUTHOR]

CRM197, which retains the same inflammatory and immune-stimulant properties as diphtheria toxin but with reduced toxicity, has been used as a safe carrier in conjugated vaccines. Expression of recombinant CRM197 in E. coli is limited due to formation of inclusion bodies. Soluble expression attempts in Bacillus subtilis, P. fluorescens, Pichia pastoris, and E. coli were partially unsuccessful or did not generate yields sufficient for industrial scale production. Multiple approaches have been attempted to produce CRM197 in E. coli, which has attractive features such as high yield, simplicity, fast growth, etc., including expression of oxidative host, concurrent expression of chaperones, or periplasmic export. Recently, alternative methods for recovery of insoluble proteins expressed in E. coli were reported. Compared to traditional denaturation/refolding, these methods used the non-denaturing solubilization agent, N-lauroylsarkosine to obtain higher recovery yields of native proteins. Based on this work, here, we focused on solubilization of CRM197 from E. coli inclusion bodies. First, CRM197 was expressed as inclusion bodies by high-level expression of recombinant CRM197 in E. coli (126.8 mg/g dcw). Then bioactive CRM197 was isolated from these inclusion bodies with high yield (108.1 mg/g dcw) through solubilization with N-lauroylsarkosine including Triton X-100 and CHAPS, and purified by Ni-affinity chromatography and size-exclusion chromatography. In this study, we present a cost-effective alternative for the production of bioactive CRM197 and compare our recovery yield with yields in other production processes. [ABSTRACT FROM AUTHOR]

Background: Inclusion of people with intellectual disability (ID) in higher postsecondary academic education is on the rise. However, there are no scientific criteria for determining the eligibility for full inclusion of students with ID in university courses. This study focuses on two models of academic inclusion for students with ID: (a) separate adapted enrichment model: students with ID study in separate enrichment courses adapted to their level; (b) full inclusion model: students with ID are included in undergraduate courses, receive academic credits and are expected to accumulate the amount of credits for a B.A. Aim: (a) To examine whether crystallized and fluid intelligence and cognitive tests can serve as screening tests for determining the appropriate placement of students with ID for the adapted enrichment model versus the full inclusion model. (b) To examine the attitudes towards the program of students with ID in the inclusion model. Method/Procedure: The sample included 31 adults with ID: students with ID who were fully included (N = 10) and students with ID who participated in the adapted enrichment model (N = 21). Crystallized and fluid intelligence were examined (WAIS-III, Wechsler, 1997) and Hebrew abstract verbal tests (Glanz, 1989). Semi-structured interviews were conducted in order to examine the attitudes of students in the inclusion model towards the program. Outcomes and results: The ANOVAs indicate that the most prominent difference between the groups was in vocabulary, knowledge and working memory. ROC analysis, a fundamental tool for diagnostic test evaluation, was used to determine the students’ eligibility for appropriate placement in the two models. Seven tests distinguished between the groups in terms of sensitivity and specificity. The interviews were analyzed according to three phases. Conclusions/Implications: The results indicate that students with ID are able to participate in undergraduate courses and achieve academic goals. The general IQ and idioms test seem to be best determiners for appropriate placement of students with ID to one of the two models. The included students with ID are motivated and self-determined in continuing in the program. [ABSTRACT FROM AUTHOR]

Lipid droplet (LD) formation occurs during infection of macrophages with numerous intracellular pathogens, including Mycobacterium tuberculosis. It is believed that M. tuberculosis and other bacteria specifically provoke LD formation as a pathogenic strategy in order to create a depot of host lipids for use as a carbon source to fuel intracellular growth. Here we show that LD formation is not a bacterially driven process during M. tuberculosis infection, but rather occurs as a result of immune activation of macrophages as part of a host defense mechanism. We show that an IFN-γ driven, HIF-1α dependent signaling pathway, previously implicated in host defense, redistributes macrophage lipids into LDs. Furthermore, we show that M. tuberculosis is able to acquire host lipids in the absence of LDs, but not in the presence of IFN-γ induced LDs. This result uncouples macrophage LD formation from bacterial acquisition of host lipids. In addition, we show that IFN-γ driven LD formation supports the production of host protective eicosanoids including PGE2 and LXB4. Finally, we demonstrate that HIF-1α and its target gene Hig2 are required for the majority of LD formation in the lungs of mice infected with M. tuberculosis, thus demonstrating that immune activation provides the primary stimulus for LD formation in vivo. Taken together our data demonstrate that macrophage LD formation is a host-driven component of the adaptive immune response to M. tuberculosis, and suggest that macrophage LDs are not an important source of nutrients for M. tuberculosis. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the effect of the inclusion of sunflower cake in the diets of lambs on meat quality. Forty male, uncastrated Santa Ines lambs with an initial average body weight of 20.9 ± 0.41 kg were used in a completely randomized design with four treatments. The lambs were fed 500 g/kg hay and 500 g/kg concentrate (corn, soybean meal, urea, ammonium sulfate and sunflower cake) in their diet, and the experimental treatments were 0, 10, 20, and 30% sunflower cake inclusion (DM basis). The inclusion of sunflower cake in the diet did not affect (P > 0.05) the content of ash and protein, overall acceptance, or sensory aroma of the lamb meat. Total lipids and moisture content of the longissimus lumborum muscle increased linearly (P ≤ 0.05) with sunflower cake inclusion. The C16:0, ΣSFA, ΣMUFA:ΣSFA ratio, Δ–9 desaturase enzyme and atherogenicity index in the longissimus lumborum muscle decreased linearly (P ≤ 0.05) with sunflower cake inclusion in lamb diets, whereas a linear increase occurred (P ≤ 0.05) in C12:0, C18:0, ΣMUFA, ΣPUFA, ΣPUFA:ΣSFA and ΣPUFA:ΣMUFA ratios, Σn–6, Σn–3, desirable fatty acids, h:H index, elongase activity, and flavor, tenderness and juiciness sensory qualities in lamb meat. Among the panelists, the highest preference (35.9%) was for meat with 30% sunflower cake inclusion in the diet. Sunflower cake up to 30% of total DM can be included in the diet of Santa Ines lambs, because lipid nutritional quality and the sensory qualities of the lamb meat increase, in addition to improvement in nutraceutical compounds, such as the CLA content, and in the AI, PUFA:SFA and PUFA:MUFA ratios, desirable fatty acids content and h:H ratio, which are beneficial to the health of consumers. [ABSTRACT FROM AUTHOR]

Plants of Schefflera actinophylla Harms. with stunted growth, chlorotic and necrotic spots and patterns, leaf epinasty and distortion are infected with impatiens necrotic spot virus classified as a member of genus Tospovirus. Studies with electron microscope revealed that in cells of S. actinophylla leaves there were no virus particle inclusions typical for tospovirus infection, but only single particles were present. The isolate of INSV was defective and the amount of INSV particles was strongly reduced in Schefflera plants. Some inclusions (I type) were composed of short branched cisterns of endoplasmic reticulum and a dark substance forming characteristic bands. The II type represented inclusions that were usually ballshaped and composed of a dark substance resembling that of the I type, but not accompanied by endoplasmic reticulum cisterns. The III type of inclusions was composed of dark, osmophilic masses. The inclusions present in the cells differ from those typical for tospovirus infection.

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11–13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates. [ABSTRACT FROM AUTHOR]

It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197–200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future. [ABSTRACT FROM AUTHOR]

The homoscleromorph sponge Oscarella carmela, first described from central California, USA is shown to represent two superficially similar but both morphologically and phylogenetically distinct species that are co-distributed. We here describe a new species as Oscarella pearsei, sp. nov. and re-describe Oscarella carmela; the original description was based upon material from both species. Further, we correct the identification of published genomic/transcriptomic resources that were originally attributed to O. carmela, and present new Illumina-sequenced transcriptome assemblies for each of these species, and the mitochondrial genome sequence for O. pearsei sp. nov. Using SSU and LSU ribosomal DNA and the mitochondrial genome, we report the phylogenetic relationships of these species relative to other Oscarella species, and find strong support for the placement of O. pearsei sp. nov. in a distinct clade within genus Oscarella defined by the presence of spherulous cells that contain paracrystalline inclusions; O. carmela lacks this cell type. Oscarella pearsei sp. nov and O. carmela can be tentatively distinguished based upon gross morphological differences such as color, surface texture and extent of mucus production, but can be more reliably identified using mitochondrial and nuclear barcode sequencing, ultrastructural characteristics of cells in the mesohyl, and the morphology of the follicle epithelium which surrounds the developing embryo in reproductively active individuals. [ABSTRACT FROM AUTHOR]

Defatted microalgal biomass derived from biorefinery can be potential feed ingredients for carnivorous fish. The present study investigated the growth, feed intake:gain and health parameters in Atlantic salmon fed for 84 days with defatted Nannochloropsis oceania as a fishmeal replacer. Fish fed feeds containing the algal biomass (at 10 and 20% inclusion, alga groups) were compared with groups that consumed alga-devoid feeds (control group). The fish that received 20% alga tended to have reduced weight gain and specific growth rate. Condition factor, feed conversion ratio and feed intake of this fish group were significantly different when compared with the control group. Hepatosomatic and viscerosomatic indices, whole body and fillet proximate composition were not affected by the dietary treatments. Digestibility of dry matter, protein, lipid, ash and energy, as well as retention of lipid and energy of the fish that received feed with 20% alga meal were also significantly different from those of the control group. Serum superoxide dismutase activity of the 10% alga-fed fish was significantly higher compared with the control fish. Although alga feeding did not cause any distal intestinal inflammation, the intestinal proteins that were altered upon feeding 20% algal meal might be pointing to systemic physiological disturbances. In conclusion, feeds with 20% alga had a negative effect on feed intake, FCR, lipid and energy retention and health of the fish. The defatted Nannochloropsis oceania can be used at modest inclusion levels, around 10%, without negative effects on the performance of Atlantic salmon. [ABSTRACT FROM AUTHOR]

Mutations in TDP-43 are associated with proteinaceous inclusions in neurons and are believed to be causative in neurodegenerative diseases such as frontotemporal dementia or amyotrophic lateral sclerosis. Here we describe a Drosophila system where we have engineered the genome to replace the endogenous TDP-43 orthologue with wild type or mutant human TDP-43(hTDP-43). In contrast to other models, these flies express both mutant and wild type hTDP-43 at similar levels to those of the endogenous gene and importantly, no age-related TDP-43 accumulation observed among all the transgenic fly lines. Immunoprecipitation of TDP-43 showed that flies with hTDP-43 mutations had increased levels of ubiquitination and phosphorylation of the hTDP-43 protein. Furthermore, histologically, flies expressing hTDP-43 M337V showed global, robust neuronal staining for phospho-TDP. All three lines: wild type hTDP-43, -G294A and -M337V were homozygous viable, with no defects in development, life span or behaviors observed. The primary behavioral defect was that flies expressing either hTDP-43 G294A or M337V showed a faster decline with age in negative geotaxis. Together, these observations implied that neurons could handle these TDP-43 mutations by phosphorylation- and ubiquitin-dependent proteasome systems, even in a background without the wild type TDP-43. Our findings suggest that these two specific TDP-43 mutations are not inherently toxic, but may require additional environmental or genetic factors to affect longevity or survival. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the C. trachomatis CT142, CT143, and CT144 proteins, which we previously showed to be type III secretion substrates. Transcriptional analyses in C. trachomatis confirmed the prediction that ct142, ct143 and ct144 are organized in an operon and revealed that their expression is likely driven by the main σ factor, σ66. In host cells infected by C. trachomatis, production of CT142 and CT143 could be detected by immunoblotting from 20–26 h post-infection. Immunofluorescence microscopy of infected cells revealed that from 20 h post-infection CT143 appeared mostly as globular structures outside of the bacterial cells but within the lumen of the inclusion. Furthermore, immunofluorescence microscopy of cells infected by C. trachomatis strains carrying plasmids producing CT142, CT143, or CT144 under the control of the ct142 promoter and with a C-terminal double hemagglutinin (2HA) epitope tag revealed that CT142-2HA, CT143-2HA or CT144-2HA showed an identical localization to chromosomally-encoded CT143. Moreover, CT142-2HA or CT144-2HA and CT143 produced by the same bacteria co-localized in the lumen of the inclusion. Overall, these data suggest that the CT142, CT143, and CT144 type III secretion substrates are secreted into the lumen of the inclusion where they might form a protein complex. [ABSTRACT FROM AUTHOR]

Background: The co-occurrence of the c.709-1G>A GRN mutation and the p.A152T MAPT variant has been identified in 18 Basque families affected by frontotemporal dementia (FTD). We aimed to investigate the influence of the p.A152T MAPT variant on the clinical and neuropathological features of these Basque GRN families. Methods and findings: We compared clinical characteristics of 14 patients who carried the c.709-1G>A GRN mutation (GRN+/A152T-) with 21 patients who carried both the c.709-1G>A GRN mutation and the p.A152T MAPT variant (GRN+/A152T+). Neuropsychological data (n = 17) and plasma progranulin levels (n = 23) were compared between groups, and 7 subjects underwent neuropathological studies. We genotyped six short tandem repeat markers in the two largest families. By the analysis of linkage disequilibrium decay in the haplotype block we estimated the time when the first ancestor to carry both genetic variants emerged. GRN+/A152T+ and GRN+/A152T- patients shared similar clinical and neuropsychological features and plasma progranulin levels. All were diagnosed with an FTD disorder, including behavioral variant FTD or non fluent / agrammatic variant primary progressive aphasia, and shared a similar pattern of neuropsychological deficits, predominantly in executive function, memory, and language. All seven participants with available brain autopsies (6 GRN+/A152T+, 1 GRN+/A152T-) showed frontotemporal lobar degeneration with TDP-43 inclusions (type A classification), which is characteristic of GRN carriers. Additionally, all seven showed mild to moderate tau inclusion burden: five cases lacked β-amyloid pathology and two cases had Alzheimer’s pathology. The co-occurrence of both genes within one individual is recent, with the birth of the first GRN+/A152T+ individual estimated to be within the last 50 generations (95% probability). Conclusions: In our sample, the p.A152T MAPT variant does not appear to show a discernible influence on the clinical phenotype of GRN carriers. Whether p.A152T confers a greater than expected propensity for tau pathology in these GRN carriers remains an open question. [ABSTRACT FROM AUTHOR]

Oils extracted from the leaves of Eupatorium adenophorum were tested in vitro and in vivo against the soilborne pathogen Pythium myriotylum which causes soft rot, a devastating disease of commercial ginger production in China. Twelve compounds accounting for 99.15% of the total oil composition were identified by GC-MS. The major components were 10Hβ-9-oxo-agerophorone (37.03%), 10Hα-9-oxo-agerophorone (37.73%) and 9-oxo-10, 11-dehydro-agerophorone (23.41%). Antifungal activity was tested by the poisoned food technique against P. myriotylum, indicating minimum inhibitory concentrations of 100μg/ml after 7 days incubation. In addition, the oil extracts greatly inhibited the formation of both wet and dry mycelial biomass. The combination of E. adenophorum oil extracts and synthetic fungicides showed a strong synergistic effect, inhibiting the mycelial growth in in vitro assays. The synergistic effect of oil extracts with fungicides could allow fungicides to be used at reduced rates in the future which has environmental advantages. Oil extracts applied at 160 and 200μg/ml concentrations to ginger rhizomes before inoculation with P. myriotylum significantly reduced the infection rate in ginger. Examination by light and transmission electron microscopy revealed that oil extracts caused swelling of the hyphae, disruption of the cell wall, degradation of the cytoplasmic organelles and shortening of the cytoplasmic inclusion. These results suggested that the plasma membrane and endomembrane systems of P. myriotylum were severely damaged by the oil extracts of E. adenophorum which offer significant potential for use as a fungicide to control P. myriotylum. [ABSTRACT FROM AUTHOR]

Diverse animal and plant viruses block the re-infection of host cells by the same or highly similar viruses through superinfection exclusion (SIE), a widely observed, yet poorly understood phenomenon. Here we demonstrate that SIE of turnip crinkle virus (TCV) is exclusively determined by p28, one of the two replication proteins encoded by this virus. p28 expressed from a TCV replicon exerts strong SIE to a different TCV replicon. Transiently expressed p28, delivered simultaneously with, or ahead of, a TCV replicon, largely recapitulates this repressive activity. Interestingly, p28-mediated SIE is dramatically enhanced by C-terminally fused epitope tags or fluorescent proteins, but weakened by N-terminal modifications, and it inversely correlates with the ability of p28 to complement the replication of a p28-defective TCV replicon. Strikingly, p28 in SIE-positive cells forms large, mobile punctate inclusions that trans-aggregate a non-coalescing, SIE-defective, yet replication-competent p28 mutant. These results support a model postulating that TCV SIE is caused by the formation of multimeric p28 complexes capable of intercepting fresh p28 monomers translated from superinfector genomes, thereby abolishing superinfector replication. This model could prove to be applicable to other RNA viruses, and offer novel targets for antiviral therapy. [ABSTRACT FROM AUTHOR]

Transactive response DNA binding protein 43 kDa (TDP-43, encoded by the TARDBP gene) is involved in transcriptional regulation and alternative splicing process. TDP-43 proteins are located in the nucleus and shuttles transcripts to the cytoplasm in normal neurons; however, they are observed in the forms of cytoplasmic inclusions in the degenerating cells. The abnormal accumulation of TDP-43 proteins is a pathologic feature of neurodegenerative diseases, such as Lou Gehrig's disease and dementia including frontotemporal lobar degeneration and Alzheimer's disease. In this study, we examined whether schizandrin, a main effective compound of Schisandra chinensis (Turcz.), so called omija in Korean, reduces TDP-43 accumulation in the hippocampal neurons in vitro. An immortalized mouse hippocampal neuronal cell line, HT22 cells, were treated with a proteasome inhibitor, MG132, in the absence or presence of schizandrin. We found that schizandrin treatment increased HT22 cell viability and reduced MG132-induced cytoplasmic accumulation of TDP-43. Our results suggest that schizandrin may be a promising compound for development of functional food materials beneficial to the neuronal protection against TDP-43 proteinopathies. [ABSTRACT FROM AUTHOR]

Reports of American shad fecundity identify two important themes regarding egg production in fishes. First, geographic variation occurs and is biologically meaningful. Shad annual fecundity decreases with increasing latitude, but predicted lifetime fecundity does not, because of a counter-gradient of survival probability, all of which can explain the adaptive significance of natal homing. Second, the appropriate method of measuring fecundity depends on the pattern of oocyte development. Historically, the relatively simple determinate-fecundity method was used; however, a recent study in a Virginia river indicates that this method may be biased, requiring the more complicated indeterminate method. We address both themes with collections from the 2015 shad spawning run in the Connecticut River, USA. Criteria for using a determinate method were satisfied for this northern population: 1) a size gap evident in the oocyte size frequency distribution, indicating group-synchronous development of yolked oocytes; 2) a decline, early in spawning, in the standing stock of yolked oocytes; and 3) low levels of atresia at the end of spawning. The determinate-method estimate of American shad annual (2015) fecundity (303,000 ± 73,400; mean ± sd) overlapped historic estimates for this and a neighboring river. The indeterminate-method estimate of annual (2015) fecundity (311,500 ± 4,500 sd) was not significantly different from the determinate-method estimate (Student’s t-test, P > 0.05). In contrast, indeterminate-method estimates of annual fecundity for a Virginia population were twice as high as that measured by the determinate method in the past. This can all be explained by fundamentally different patterns of oogenesis (i.e., group synchrony versus asynchrony with respect to yolk development) at different latitudes. American shad, which is distributed within its native range from the Canadian maritimes to Florida, USA (50–30°N), may be particularly well suited to evaluate intra-specific variation in oocyte development, a relatively unexplored life history trait. [ABSTRACT FROM AUTHOR]

The scorpion has been extensively used in various pharmacological profiles or as food supplies. The exploration of scorpion venom has been reported due to the presence of recombinant peptides. rBmαTX14 is an α-neurotoxin extracted from the venom gland of the East Asian scorpion Buthus martensii Karsch and can affect ion channel conductance. Here, we investigated the functions of rBmαTX14 using the Caenorhabditis elegans model. Using western blot analysis, rBmαTX14 was shown to be expressed both in the cytoplasm and inclusion bodies in the E.coli Rosetta (DE3) strain. Circular dichroism spectroscopy analysis demonstrated that purified rBmαTX14 retained its biological structures. Next, feeding nematodes with E.coli Rosetta (DE3) expressing rBmαTX14 caused extension of the life span and promoted the locomotion of the nematodes. In addition, we identified several genes that play various roles in the life span and locomotion of C. elegans through microarray analysis and quantitative real-time PCR. Furthermore, if the amino acid site H15 of rBmαTX14 was mutated, rBmαTX14 no longer promoted the C. elegans life span. In conclusion, the results not only demonstrated the functions and mechanism of rBmαTX14 in C. elegans, but also provided the new sight in the utility of recombinant peptides from scorpion venom. [ABSTRACT FROM AUTHOR]

Fabry nephropathy is associated with progressive accumulation of globotriaosylceramide (GL3) in podocytes. Reducing this GL3 burden may reduce podocyte injury. Sensitive methods to quantify podocyte GL3 content may determine whether a given strategy can benefit podocytes in Fabry disease. We developed an unbiased electron microscopic stereological method to estimate the average volume of podocytes and their GL3 inclusions in 6 paired pre- and post-enzyme replacement therapy (ERT) biopsies from 5 men with Fabry disease. Podocyte GL3 content was regularly reduced (average 73%) after 11–12 months of ERT. This was not detectable using a semi-quantitative approach. Parallel to GL3 reduction, podocytes became remarkably smaller (average 63%). These reductions in podocyte GL3 content or size were not significantly correlated with changes in foot process width (FPW). However, FPW after ERT was significantly correlated with the magnitude of the decrease in podocyte GL3 content from baseline to 11–12 months of ERT. Also podocytes exocytosed GL3 inclusions, a phenomenon correlated with their reduction in their GL3 content. Demonstrable after11–12 months, reduction in podocyte GL3 content allows for early assessment of treatment efficacy and shorter clinical trials in Fabry disease. [ABSTRACT FROM AUTHOR]

The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be invaluable for investigations into gene regulation and function. [ABSTRACT FROM AUTHOR]

Autophagy plays a major role in the elimination of cellular waste components, the renewal of intracellular proteins and the prevention of the build-up of redundant or defective material. It is fundamental for the maintenance of homeostasis and especially important in post-mitotic neuronal cells, which, without competent autophagy, accumulate protein aggregates and degenerate. Many neurodegenerative diseases are associated with defective autophagy; however, whether altered protein turnover or accumulation of misfolded, aggregate-prone proteins is the primary insult in neurodegeneration has long been a matter of debate. Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by selective degeneration of motor neurons. Most of the ALS cases occur in sporadic forms (SALS), while 10%-15% of the cases have a positive familial history (FALS). The accumulation in the cell of misfolded/abnormal proteins is a hallmark of both SALS and FALS, and altered protein degradation due to autophagy dysregulation has been proposed to contribute to ALS pathogenesis. In this review, we focus on the main molecular features of autophagy to provide a framework for discussion of our recent findings about the role in disease pathogenesis of the ALS-linked form of the VAPB gene product, a mutant protein that drives the generation of unusual cytoplasmic inclusions. [ABSTRACT FROM AUTHOR]

The ferrimagnetic mineral magnetite is biomineralized by magnetotactic microorganisms and a diverse range of animals. Here we demonstrate that confocal Raman microscopy can be used to visualize chains of magnetite crystals in magnetotactic bacteria, even though magnetite is a poor Raman scatterer and in bacteria occurs in typical grain sizes of only 35–120 nm, well below the diffraction-limited optical resolution. When using long integration times together with low laser power (<0.25 mW) to prevent laser induced damage of magnetite, we can identify and map magnetite by its characteristic Raman spectrum (303, 535, 665 ) against a large autofluorescence background in our natural magnetotactic bacteria samples. While greigite (cubic ; Raman lines of 253 and 351 ) is often found in the Deltaproteobacteria class, it is not present in our samples. In intracellular sulfur globules of Candidatus Magnetobacterium bavaricum (Nitrospirae), we identified the sole presence of cyclo-octasulfur (: 151, 219, 467 ), using green (532 nm), red (638 nm) and near-infrared excitation (785 nm). The Raman-spectra of phosphorous-rich intracellular accumulations point to orthophosphate in magnetic vibrios and to polyphosphate in magnetic cocci. Under green excitation, the cell envelopes are dominated by the resonant Raman lines of the heme cofactor of the b or c-type cytochrome, which can be used as a strong marker for label-free live-cell imaging of bacterial cytoplasmic membranes, as well as an indicator for the redox state. [ABSTRACT FROM AUTHOR]