Nuclear Fraction Research Articles

Low abundance of insulin-induced gene 1 contributes to SREBP-1c processing and hepatic steatosis in dairy cows with severe fatty liver

Fatty liver is a major metabolic disorder of high-producing dairy cows during the transition period. In nonruminants, it is well established that insulin-induced gene 1 (INSIG1) plays a crucial role in regulating hepatic lipogenesis by controlling the anchoring of sterol regulatory element-binding protein 1 (SREBP-1) on the endoplasmic reticulum along with SREBP cleavage-activating protein (SCAP). Whether the INSIG1-SCAP-SREBP-1c transport axis is affected in cows experiencing fatty liver is unknown. Thus, the aim of this study was to investigate the potential role of INSIG1-SCAP-SREBP-1c axis in the progression of fatty liver in dairy cows. For in vivo experiments, 24 dairy cows at the start of their fourth lactation (median; range 3-5) and 8 d in milk (median; range 4-12 d) were selected into a healthy group [n = 12; triglyceride (TG) content <1%] and a severe fatty liver group (n = 12; TG content >10%) according to their hepatic TG content. Blood samples were collected for detecting serum concentrations of free fatty acids, β-hydroxybutyrate, and glucose. Compared with healthy cows, cows with severe fatty liver had higher serum concentrations of β-hydroxybutyrate and free fatty acids and lower concentration of glucose. Liver biopsies were used to detect the status of INSIG1-SCAP-SREBP-1c axis, and the mRNA expression of SREBP-1c-target lipogenic genes acetyl-CoA carboxylase α (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1). Cows with severe fatty liver had lower protein expression of INSIG1 in the hepatocyte endoplasmic reticulum fraction, greater protein expression of SCAP and precursor SREBP-1c in the hepatocyte Golgi fraction, and greater protein expression of mature SREBP-1c in the hepatocyte nuclear fraction. In addition, the mRNA expression of SREBP-1c-target lipogenic genes ACACA, FASN, and DGAT1 was greater in the liver of dairy cows with severe fatty liver. In vitro experiments were conducted on hepatocytes isolated from 5 healthy 1-d-old female Holstein calves, and hepatocytes from each calf were run independently. First, hepatocytes were treated with 0, 200, or 400 μM palmitic acid (PA) for 12 h. Exogenous PA treatment decreased INSIG1 protein abundance, enhanced the endoplasmic reticulum to Golgi export of SCAP-precursor SREBP-1c complex and the nuclear translocation of mature SREBP-1c, all of which was associated with increased transcriptional activation of lipogenic genes and TG synthesis. Second, hepatocytes were transfected with INSIG1-overexpressing adenovirus for 48 h and treated with 400 μM PA 12 h before the end of transfection. Overexpressing INSIG1 inhibited PA-induced SREBP-1c processing, upregulation of lipogenic genes, and TG synthesis in hepatocytes. Overall, the present in vivo and in vitro results indicated that the low abundance of INSIG1 contributed to SREBP-1c processing and hepatic steatosis in dairy cows. Thus, the INSIG1-SCAP-SREBP-1c axis may be a novel target for treatment of fatty liver in dairy cows.

Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis.

Lentinan (LNT) isolated from Lentinus edodes is a vital host defense potentiator previously utilized as an adjuvant in cancer therapy. The present study investigated the effect of LNT on the mouse hepatocellular carcinoma (HCC) cell line Hepa1‑6 and its possible mechanism. Mouse HCC apoptosis and its potential associated mechanism were then explored using invitro and invivo approaches. Forinvitroapproaches, the effect of LNT on the proliferation of Hepa1‑6 cells was investigated by Cell Counting Kit‑8 assay. Annexin V‑FITC staining and flow cytometry were applied to explore HCC apoptosis. Western blotting was used to analyze related proteins, such as EGR1, phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (p‑Akt), protein kinase B (Akt), B lymphocyte‑2 (Bcl‑2), Bcl2 family‑associated X protein (Bax), etc. Cellular immunofluorescence staining was employed to assess the localization and expression of EGR1 and PTEN in nuclear and cytoplasmic fractions of Hepa1‑6 cells. The association betweenEGR1andPTENwas explored by EGR1 overexpressionin cell lines. Forinvivomethods, a mouse model of diethylnitrosamine (DEN)‑induced primary liver cancer was established using C57BL/6 mice to investigate the inhibitory effect of LNT on liver cancer. Histopathology of liver tissue from mice was detected by hematoxylin‑eosin staining and immunohistochemical assay. Invitro and invivo results showed that LNT can inhibit the proliferation and promote the apoptosis of mouse HCC cells. Besides, LNT increased the expression of EGR1 in Hepa1‑6 cells, which is translocated to the nucleus to function as a transcriptional factor. EGR1 then activates the expression of the tumor suppressor PTEN, thereby inhibiting the activation of the AKT signaling pathway. These data revealed a novel anti‑tumor mechanism by which LNT can induce apoptosis to inhibit mouse HCC progression through the EGR1/PTEN/AKT axis. These results provide a scientific basis for the potential use of LNT in drug development and clinical applications associated with primary liver cancer.

Repeat RNA structure and protein interactions influence RAN translation and neurodegenerative phenotypes in models of FTD and ALS

AbstractBackgroundThe most common genetic cause of Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) is an intronic GGGGCC hexanucleotide repeat expansion in C9orf72 (C9FTD/ALS). Transcribed GGGGCC repeat RNAs sequester RNA‐binding proteins (RBPs) into nuclear and cytoplasmic foci, which inhibits their normal functions. GGGGCC repeat RNAs also support translation of toxic repeat peptides through a process known as repeat associated non‐AUG (RAN) translation. Here we sought to understand how repeat RNA secondary structure and RBP engagements that occur during RNA transcription, processing, and trafficking influence GGGGCC RAN translation and repeat elicited toxicity, with a goal of identifying novel therapeutic targets.MethodWe developed a cellular RNA‐capture methodology coupled to mass spectrometry to identify GGGGCC repeat RNA‐interacting proteins within specific cellular compartments. In parallel, we used chemical approaches to probe GGGGCC repeat RNA secondary structures inside cells and RAN translation assays to determine whether repeat RNA secondary structures and/or interacting RBPs influence RAN translation efficiency. We employed Drosophila and neuronal models of repeat expansions to evaluate potential RBPs as disease modifiers.ResultOur RNA structural studies revealed that GGGGCC repeat RNAs form primarily hairpin structures, rather than G‐quadruplex structures, in cells. Folding repat RNAs into such hairpins supports RAN translation whereas folding repeat RNAs into g‐quadruplex structures strongly inhibits their translation. We identified multiple RBPs enriching in the nuclear fraction of GGGGCC RNA‐interactome, including hnRNPH1 and SR (serine/arginine‐rich domain) proteins, which are known components of the nuclear foci found in C9FTD/ALS patient cells. Our comparative analysis found that lowering SR protein expression or modifying its phosphorylation modified toxicity in Drosophila models of multiple repeat expansion disorders. Currently we are investigating how repeat RNA structures influence RBP interactions and whether structure dependent RBP interactions selectively modulate RAN translation.ConclusionWe present a comprehensive approach to evaluate the roles of repeat RNA secondary structures and critical RBPs in RNA toxicity underlying dementia‐associated repeat expansion disorders.

Oxidative stress responses and their alterations in the Nrf2-NMDA receptor pathway in the brain of suicide victims.

Suicide is a global public health concern. There is evidence of an association between suicidal behavior and depressive disorders (DDs). An increasing number of studies have suggested that nuclear factor erythroid-derived 2-like 2 (Nrf2), a major endogenous regulator of the oxidative stress response, can be a novel target for the neurobiology of suicide-related disorders (including depression). This study aimed to investigate the relationship between oxidative stress progression, Nrf2 regulation, and N-methyl-D-aspartate receptor (NMDA) subunit composition in the hippocampus (Hp) and frontal cortex (FCx) of suicide victims (n=14) and matched controls (n=8). Furthermore, zinc and magnesium concentrations and their potency to inhibit [3H] MK-801 (radioactively labeled form of MK-801 - dizocilpine, a well-characterized NMDAR channel uncompetitive antagonist frequently used in receptor-binding assays) binding to NMDA receptor channels were measured. Our results revealed a statistically significant increase in protein carbonyl levels and thiobarbituric acid-reactive substances (TBARS) concentrations in Hp and FCx of suicide victims. Enhanced superoxide dismutase (SOD) activity (only in FCx) in suicides compared to controls was shown. These alterations were accompanied by an increase in Nrf2 protein levels in whole homogeneous tissue lysates and cytosolic fractions of Hp and FCx. Importantly, suicide victims presented a significant reduction in Nrf2 protein levels in the nuclear fraction of FCx. Finally, the observed decrease in N-methyl-D-aspartate receptor subunit 2B (GluN2B) and postsynaptic density proteins 95 (PSD-95) protein levels was associated with a statistically significant reduction in magnesium levels in the FCx of suicide victims. These results confirm for the first time that increased oxidative stress parameters are related to Nrf2 protein changes and alterations in the NMDA receptor complex in the pathophysiology of suicidal behavior.

αCGRP deficiency aggravates pulmonary fibrosis by activating the PPARγ signaling pathway

In order to explore whether αCGRP (Calca) deficiency aggravates pulmonary fibrosis (PF). Clinical data from patients with PF (n = 52) were retrospectively analyzed. Lung tissue from a bleomycin (BLM)-induced rat model was compared with that of Calca-knockout (KO) and wild type (WT) using immunohistochemistry, RNA-seq, and UPLC-MS/MS metabolomic analyses. The results showed that decreased αCGRP expression and activation of the type 2 immune response were detected in patients with PF. In BLM-induced and Calca-KO rats, αCGRP deficiency potentiated apoptosis of AECs and induced M2 macrophages. RNA-seq identified enrichment of pathways involved in nuclear translocation and immune system disorders in Calca-KO rats compared to WT. Mass spectrometry of lung tissue from Calca-KO rats showed abnormal lipid metabolism, including increased levels of LTB4, PDX, 1-HETE. PPAR pathway signaling was significantly induced in both transcriptomic and metabolomic datasets in Calca-KO rats, and immunofluorescence analysis confirmed that the nuclear translocation of PPARγ in BLM-treated and Calca-KO rats was synchronized with STAT6 localization in the cytoplasmic and nuclear fractions. In conclusion, αCGRP is protective against PF, and αCGRP deficiency promotes M2 polarization of macrophages, probably by activating the PPARγ pathway, which leads to activation of the type 2 immune response and accelerates PF development.

Red mud waste/nanoclay/polystyrene‐modified epoxy hybrid composites: Mechanical, thermal, and flammability properties

AbstractIn this study, bisphenol A‐type epoxy (ER) and epoxy phenol novolac resins (EPN) with two amine‐type curing agents (KH 816 and IPOX EH 2041) having different total amine values were selected. The mechanical properties and density of the EPN resin were found higher in the case of both curing agents. EPN was modified with polystyrene (EPN‐PS) and used as a matrix using an IPOX hardener. Nano‐ and hybrid composites were prepared with pristine and modified with tetramethylammonium chloride nano montmorillonite (NC and MNC), and red mud waste (RMW). The chemical structure was elucidated by the Fourier transform infrared (FT‐IR). The composites were characterized by scanning electron microscopy and x‐ray diffraction. Appropriate PS and NC ratio was accepted as 4% and 2% by weight, respectively. Curing degrees calculated from the FT‐IR spectra of ER, EPN, and EPN‐PS were 99.3%, 99.9%, and 86.5%, respectively. A comparative study of the two binary systems; (i) 2 wt% NC‐(15–35) wt% RMW; and (ii) 2 wt% of MNC‐(15–35) wt% of RMW on the mechanical, thermal, and flammability properties of EPN‐PS matrix was carried out. The appropriate RMW ratio was 30 wt%. Hybrid composites with 25–30 wt% RMW can be considered self‐extinguishing materials.

Museomics and phylogenomics of lovebirds (Psittaciformes, Psittaculidae, Agapornis) using low-coverage whole-genome sequencing

Natural history collections contain specimens that provide important insights into studies of ecology and evolution. With the advancement of high-throughput sequencing, historical DNA (hDNA) from museum specimens has become a valuable source of genomic data to study the evolutionary history of organisms. Low-coverage whole genome sequencing (WGS) has been increasingly applied to museum specimens for analyzing organelle genomes, but is still uncommon for genotyping the nuclear DNA fraction. In this study, we applied low-coverage WGS to phylogenomic analyses of parrots in the genus Agapornis by including both modern samples and historical specimens of ∼100-year-old. Agapornis are small-sized African and Malagasy parrots with diverse characters. Earlier phylogenetic studies failed to resolve the positions of some key lineages, prohibiting a robust interpretation of the biogeography and evolution of these African parrots. Here, we demonstrated the use of low-coverage WGS for generating both mitochondrial and nuclear genomic data, and evaluated data quality differences between modern and historical samples. Our resolved Agapornis phylogeny indicates the ancestor of Agapornis likely colonized Madagascar from Australasia by trans-oceanic dispersal events before dispersing to the African continent. Genome-wide SNPs also allowed us to identify the parental origins of hybrid Agapornis individuals. This study demonstrates the potential of applying low-coverage WGS to phylogenomics and population genomics analyses and illustrates how including historical museum specimens can address outstanding questions regarding the evolutionary history of contemporary lineages.

Gender-based cytomorphometric analysis of buccal exfoliated cells in a South Indian population.

To assess differences based on gender in nuclear area (NA), cytoplasmic area (CA), and nuclear-cytoplasmic ratio (N:C ratio) values for clinically normal buccal smears in apparently healthy subjects of a South Indian population, thereby providing definitive baseline cytomorphometric measurements for the South Indian population. Buccal smears were collected from 60 healthy subjects (30 males and 30 females) of a South Indian population above 18 years in age. Values of the NA and CA were measured, and the N:C ratio calculated, using ImageJ software. Statistical analysis of the data with independent t tests and 95% confidence intervals was done using SPSS version 21 (significance set at P < 0.05). The results showed that there were significant differences in the NA, CA, and N:C values between males and females irrespective of age (P = 0.01). Definitive gender-specific baseline cytomorphometric data can be established using exfoliative cytology for the South Indian population, and this may be helpful in deciphering the occurrence of oral pre-malignant diseases and oral squamous cell carcinoma in this population, as the incidence of these conditions varies with gender and different ethnic groups.

Species specifics of morphology of the liver of the fishes of the Cyprinidae family

Providing mankind with high-quality products of aquaculture is possible only by introduction of modern industrial technologies to fish farming, growing fish based on modern scientific achievements. Assessment of the ecotoxic situation and identification of impacts of various unfavourable factors of aquatic environment on aquatic organisms should be made through morphological studies of the organs that are first to encounter the impact. The study revealed species specifics of the morphology of the liver of the fishes of the Cyprinidae family – Prussian carp (Carassius gibelio), Eurasian carp (Cyprinus carpio), and bighead carp (Hypophthalmichthys nobilis), which vary by extent of the motor activity in the aquatic environment, nutrition, etc. We determined that during phylogenetic development of fish that grow in the aquatic environment, there occurs a certain restructuring of the liver: the adaptation to various living conditions were accompanied by changes in a number of parameters of macro- and microscopic architectonics of the liver. Cyprinus carpio and Carassius gibelio (omnivores) have a two-lobe liver, while Hypophthalmichthys nobilis (a herbivore) has a three-lobe liver. For C. carpio and H. nobilis, a characteristic feature of the liver was presence of the hepatopancreatitis (the liver and the pancreas, associated into a single organ), and in C. gibelio, they are differentiated into individual organs. A peculiarity of the microscopic structure of the liver of the Cyprinidae family is poorly developed interlobular connective tissue, and parenchyma of the liver lobule has a tubular structure as polyhedral, curved thick-walled tubules, the walls of which are hepatocytes. The greatest amounts of cytoplasm and karyoplasms were seen in C. gibelio, equaling respectively 12.98 ± 1.42 and 0.40 ± 0.02 µm³. The lowest volume of the indicated parameters was in C. carpio, particularly 2.97 ± 0.22 and 0.21 ± 0.01 µm³, respectively. The lowest nuclear-cytoplasmic ratio was observed in hepatocytes of C. gibelio (0.0316 ± 0.0024). The conducted morphological studies at the levels of organs, tissues and cells can reveal how the fishes’ bodies adapt to particular living conditions and impacts of environmental factors.

Butyrate modulation of inflammatory responses to lipopolysaccharide in porcine monocytes

Abstract Butyrate is a short-chain fatty acid produced through microbial fermentation of dietary fibers in the lower intestinal tract. Butyrate production is associated with enhanced mucosal immunity, increased barrier integrity, and modulation of immune cells. Butyrate driven immunomodulation occurs through a variety of mechanisms, including histone deacetylase (HDAC) inhibitor activity and inhibition of NF-kB signaling. Butyrate impacts lipopolysaccharide (LPS)-induced cytokine production and gene expression by porcine monocytes in a dose-dependent manner. To investigate the mechanisms driving butyrate modulation of porcine monocyte responses to LPS, peripheral monocytes were cultured with LPS +/− butyrate. After incubation, cells were processed for collection of cytoplasmic and nuclear fractions. HDAC activity in nuclear fractions and NF-kB signaling state (IkBa, p-IkBa, p65, p-p65) in cytoplasmic fractions was assessed. Butyrate did not inhibit HDAC activity after 1 hour (h) but at 24 h, butyrate exposure resulted in inhibition of HDAC activity regardless of LPS exposure, in a dose-dependent manner. Neither the low nor high concentration of butyrate altered IkBa, p-IkBa, p65, or p-p65 status, though p-IkBa and p-p65 status was dependent on LPS exposure. Overall, the impact of butyrate on HDAC activity was time and dose-dependent, and butyrate had little to no effect on NF-kB signaling induced by LPS exposure. Continued investigation is warranted to understand the potential mechanisms driving butyrate modulation of monocyte responses to LPS to enhance abilities to modulate immune status. USDA-ARS CRIS 005

EFFECTS OF COLD SHOCK ON THE SIZE AND ACTIVITY OF &lt;i&gt;SCORPAENA PORCUS&lt;/i&gt; (LINNAEUS, 1758) NUCLEAR ERYTHROCYTES (&lt;i&gt;IN VITRO&lt;/i&gt; EXPERIMENTS)

The in vitro effect of cold shock on erythrocytes of the benthic scorpionfish Scorpaena porcus L. was studied. The blood samples were exposed to 22°С (control group) and 4°С (experimental group) for 3 hours. Cold shock had no significant impact on erythrocyte scorpionfish vital function. The cells maintained the integrity of cytoplasmic membranes, mitochondrial potential and the level of oxidative processes, that the constant values of Propidium Iodide (PI), Rhodamine 123 (R123) and 2',7'-Dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence levels demonstrated. Main changes referred to erythrocyte shape and nucleus functional activity. Cells gained more roundish shape, nucleus size increased with the growth of the nuclear-cytoplasmic ratio (NCR) and the decrease of fluorescence intensity of the cells labeled with SYBR Green I. It appears to be related to the activation of transcriptomic processes.

Morphological Analysis of the Hemolymph Cell Composition in the Bivalve Mollusk &lt;i&gt;Anadara&lt;/i&gt; &lt;i&gt;broughtonii&lt;/i&gt; Schrenck, 1867 (Sea of Japan)

The hemolymph cells of the ark clam Anadara broughtonii were examined using light microscopy, flow cytometry and gradient centrifugation. All three methods of analysis made it possible to identify two main types of cells in the hemolymph of the ark clam Anadara broughtonii – large granular erythrocytes and small agra-nular amebocytes. Erythrocytes accounted for 95.6 ± 0.9% of the total number of hemolymph cells. Erythrocytes were hemoglobin-containing cells with a great number of granules in the cytoplasm, a low nuclear-cytoplasmic ratio (NCR) and a lower intensity of cellular respiration compared to amebocytes. Amebocytes are cells of predominantly irregular shape with a high number of cells that do not contain or contain no more than 10 granular inclusions in the cytoplasm. All types of hemocytes found in the ark clam hemolymph demonstrated the same ability to spontaneously produce reactive oxygen species. For the first time, it has been shown that red blood cells of the ark clam Anadara broughtonii are capable of phagocytosis. At the same time, amebocytes absorbed on the average two times more zymosan particles (10.3 ± 0.7 pcs.), compared to erythrocytes (5.3 ± 0.1 pcs.).

Variance effective population size is affected by census size in sub-structured populations.

Measurement of allele frequency shifts between temporally spaced samples has long been used for assessment of effective population size (Ne ), and this 'temporal method' provides estimates of Ne referred to as variance effective size (NeV ). We show that NeV of a local population that belongs to a sub-structured population (a metapopulation) is determined not only by genetic drift and migration rate (m), but also by the census size (Nc ). The realized NeV of a local population can either increase or decrease with increasing m, depending on the relationship between Ne and Nc in isolation. This is shown by explicit mathematical expressions for the factors affecting NeV derived for an island model of migration. We verify analytical results using high-resolution computer simulations, and show that the phenomenon is not restricted to the island model migration pattern. The effect of Nc on the realized NeV of a local subpopulation is most pronounced at high migration rates. We show that Nc only affects local NeV , whereas NeV for the metapopulation as a whole, inbreeding (NeI ), and linkage disequilibrium (NeLD ) effective size are all independent of Nc . Our results provide a possible explanation to the large variation of Ne /Nc ratios reported in the literature, where Ne is frequently estimated by NeV . They are also important for the interpretation of empirical Ne estimates in genetic management where local NeV is often used as a substitute for inbreeding effective size, and we suggest an increased focus on metapopulation NeV as a proxy for NeI .

Differentiation Disorders of Chara vulgaris Spermatids following Treatment with Propyzamide.

Microtubules are cytoskeletal cell elements that also build flagella and cilia. Moreover, these structures participate in spermatogenesis and form a microtubular manchette during spermiogenesis. The present study aims to assess the influence of propyzamide, a microtubule-disrupting agent, on alga Chara vulgaris spermatids during their differentiation by means of immunofluorescent and electron microscopy methods. Propyzamide blocks the functioning of the β-tubulin microtubule subunit, which results in the creation of a distorted shape of a sperm nucleus at some stages. Present ultrastructural studies confirm these changes. In nuclei, an altered chromatin arrangement and nuclear envelope fragmentation were observed in the research as a result of incorrect nucleus-cytoplasm transport behavior that disturbed the action of proteolytic enzymes and the chromatin remodeling process. In the cytoplasm, large autolytic vacuoles and the dilated endoplasmic reticulum (ER) system, as well as mitochondria, were revealed in the studies. In some spermatids, the arrangement of microtubules present in the manchette was disturbed and the structure was also fragmented. The observations made in the research at present show that, despite some differences in the manchette between Chara and mammals, and probably also in the alga under study, microtubules participate in the intramanchette transport (IMT) process, which is essential during spermatid differentiation. In the present study, the effect of propyzamide on Chara spermiogenesis is also presented for the first time; however, the role of microtubule-associated proteins in this process still needs to be elucidated in the literature.

Arylalkylamine N-acetyltransferase (AANAT): Blue light induction, nuclear translocation, and potential role in the survival of chicken retina neuronal cells.

In vertebrates, arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is the time-keeping and key regulatory enzyme in melatonin (Mel) biosynthesis. AANAT is present in the pineal gland, retina, and other regions where it is controlled by light, cyclic adenosine monophosphate (cAMP) levels, and the molecular clock. AANAT converts serotonin to N-acetyl serotonin (NAS) and the last enzyme in the pathway, hydroxy-o-methyltransferase (HIOMT), forms Mel by NAS methylation. We have previously shown that AANAT is expressed in chicken retinal ganglion cells (RGCs) during daytime at the level of mRNA and enzyme activity. Here we investigated the presence of AANAT protein and mRNA throughout development in the chicken embryonic retina as well as AANAT expression, phosphorylation, and its sub-cellular localization in primary cultures of retinal neurons from E10 embryonic retinas exposed to blue light (BL) and controls kept in the dark (D). From embryonic days 7-10 (E7-10) AANAT mRNA and protein were visualized mainly concentrated in the forming ganglion cell layer (GCL), while from E17 through postnatal days, expression was detectable all through the different retinal cell layers. At postnatal day 10 (PN10) when animals were subjected to a 12:12 h LD cycle, AANAT was mainly expressed in the GCL and inner nuclear layer cells at noon (Zeitgeber Time (ZT 6)) and in the photoreceptor cell layer at night (ZT 21). Primary cultures of retinal neurons exhibited an induction of AANAT protein when cells were exposed to BL for 1 h as compared with D controls. After BL exposure, AANAT showed a significant change in intracellular localization from the cytoplasm to the nucleus in the BL condition, remaining in the nucleus 1-2 h in the D after BL stimulation. BL induction of nuclear AANAT was substantially inhibited when cultures were treated with the protein synthesis inhibitor cycloheximide (CHD). Furthermore, the phosphorylated form of the enzyme (pAANAT) increased after BL in nuclear fractions obtained from primary cultures as compared with D controls. Finally, the knockdown of AANAT by sh-RNA in primary cultures affected cell viability regardless of the light condition. AANAT knockdown also affected the redox balance, sh-AANAT treated cultures showing higher levels of reactive oxygen species (ROS) than in the sh-control. Our results support the idea that AANAT is a BL-sensing enzyme in the inner retina of diurnal vertebrates, undergoing phosphorylation and nuclear importation in response to BL stimulation. Moreover, it can be inferred that AANAT plays a novel role in nuclear function, cell viability, and, likely, through redox balance regulation.

Mitotic chromosomes scale to nuclear-cytoplasmic ratio and cell size in Xenopus.

During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to scale coordinately with mitotic spindles, but the underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle, and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.

Knockdown of BAP31 Overcomes Hepatocellular Carcinoma Doxorubicin Resistance through Downregulation of Survivin.

The expression of B-cell receptor associated protein 31 (BAP31) is increased in many tumor types, and it is reported to participate in proliferation, migration, and apoptosis. However, the relationship between BAP31 and chemoresistance is uncertain. This study investigated the role of BAP31 in regulating the doxorubicin (Dox) resistance of hepatocellular carcinoma (HCC). The expression of proteins was assessed by Western blotting. The correlation between BAP31 expression and Dox resistance was examined by MTT and colony formation assays. Apoptosis was analyzed by flow cytometry and TdT-mediated dUTP nick end labeling assays. Western blot and immunofluorescence analyses were performed in the knockdown cell lines to explore the possible mechanisms. In this study, BAP31 was strongly expressed, and knockdown of BAP31 increased Dox chemosensitivity in cancer cells. Furthermore, the expression of BAP31 was higher in the Dox-resistant HCC cells than that in their parental cells; knockdown of BAP31 reduced the half maximal inhibitory concentration value and overcame Dox resistance in Dox-resistant HCC cells. In HCC cells, knockdown of BAP31 increased Dox-induced apoptosis and enhanced Dox chemosensitivity in vitro and in vivo. The potential mechanism by which BAP31 increased Dox-induced apoptosis is that BAP31 inhibited survivin expression by promoting FoxO1 nucleus-cytoplasm translocation. Knockdown of BAP31 and survivin had a synergistic effect on Dox chemosensitivity by enhancing the apoptosis of HCC cells. These findings reveal that BAP31 knockdown enhances Dox chemosensitivity through the downregulation of survivin, suggesting that BAP31 is a potential therapeutic target for improving the treatment response of HCC with resistance to Dox.

Computer analysis of abnormal proliferation and transformation cells in gastric mucosa and its clinical significance

ABSTRACT To investigate the image computer analysis of abnormally proliferating transformed cells of gastric mucosa and its clinical significance. The pathological pictures of gastric adenomatous polyp cells, abnormally proliferating altered cells, and tubular adenocarcinoma cells in the stomach mucosa were assessed by image computer method on a total of 96 gastroscopic biopsy and ESD resection specimens. The data of cytoplasmic area, nuclear area, nuclear-cytoplasmic ratio, nuclear factor and N-heterotypic index of gastric adenomatous polyps, abnormal proliferative transformation and gastric intramucosal tubular adenocarcinoma were collected, and the mean, standard deviation and variance were calculated respectively. Standard Error, Maximum, Minimum Parameters and Statistical Structure. There were substantial discrepancies between gastric mucosal gastric adenomatous polyp cells and gastric mucosal abnormally proliferating transformed cells, according to the five data in the abnormal cells in the stomach mucosal proliferation area (p < 0.01); There was no significant difference between cells (p > 0.05). Computer analysis of cell images can provide quantitative values for the pathological diagnosis of gastric adenomatous polyp cells, abnormally proliferating transformed cells and tubular adenocarcinoma cells in the gastric mucosa, especially the degree of atypical proliferation. The monitoring of abnormally proliferated and transformed cells in gastric mucosa is of great significance for clinicians to accurately treat and track cell transformation, and to control the occurrence and development of gastric adenocarcinoma.

Cytological structure of a vaginal stroke in American mink during estrus

The article presents the data of domestic and foreign authors on the features of the oestrous process of American minks. The results of studies of the cytological structure of the vaginal smear in American minks during estrus are presented. The study was conducted on clinically healthy American mink females of the Standard dark brown (+/+) genotype in 92 animals before the technological rut (February 20-28). A vaginal smear was taken at different periods of changes in the external genital organs - the genital loop, taking into account the scoring of its development (from 0 to 5 points). Microscopic examination of smears taken during this period showed a predominance of superficial, intermediate, and parabasal cells and a small number of basal and spindle cells. The analysis of the nuclear-cytoplasmic ratio made it possible to indicate the specificity of the morphometric parameters of the cells of the vaginal integumentary epithelium. Surface epitheliocytes and intermediate cells have a sizeable cytoplasmic area and a low nuclear-cytoplasmic ratio of 0.034. Parabasal cells have the minimum size of the nucleus and cytoplasm but a high value of the nuclear-cytoplasmic percentage, seven times higher than that of superficial cells and three times intermediate. Quantitative proportions of epithelial cells of the vaginal smear cytogram from February 20 to 28 correspond to the proestrus-estrus stage. The data obtained indicate significant features of the morphological changes in the integumentary epithelium of the vagina and the difference in the cytological dynamics of the oestrous cycle in American minks from other mammals. The cytogram of the vaginal smear of minks with a 4-5 point assessment of the readiness of females for mating does not contain the predominant number of keratinized epithelial cells, which is characteristic of most mammals during the period of ovulation. An important morphological criterion in the analysis of the vaginal smear cytogram in minks is the value of the ratio of superficial and parabasal cells and scales and parabasal cells.

Mitochondrial, cell cycle control and neuritogenesis alterations in an iPSC-based neurodevelopmental model for schizophrenia.

Schizophrenia is a severe psychiatric disorder of neurodevelopmental origin that affects around 1% of the world's population. Proteomic studies and other approaches have provided evidence of compromised cellular processes in the disorder, including mitochondrial function. Most of the studies so far have been conducted on postmortem brain tissue from patients, and therefore, do not allow the evaluation of the neurodevelopmental aspect of the disorder. To circumvent that, we studied the mitochondrial and nuclear proteomes of neural stem cells (NSCs) and neurons derived from induced pluripotent stem cells (iPSCs) from schizophrenia patients versus healthy controls to assess possible alterations related to energy metabolism and mitochondrial function during neurodevelopment in the disorder. Our results revealed differentially expressed proteins in pathways related to mitochondrial function, cell cycle control, DNA repair and neuritogenesis and their possible implication in key process of neurodevelopment, such as neuronal differentiation and axonal guidance signaling. Moreover, functional analysis of NSCs revealed alterations in mitochondrial oxygen consumption in schizophrenia-derived cells and a tendency of higher levels of intracellular reactive oxygen species (ROS). Hence, this study shows evidence that alterations in important cellular processes are present during neurodevelopment and could be involved with the establishment of schizophrenia, as well as the phenotypic traits observed in adult patients. Neural stem cells (NSCs) and neurons were derived from induced pluripotent stem cells (iPSCs) from schizophrenia patients and controls. Proteomic analyses were performed on the enriched mitochondrial and nuclear fractions of NSCs and neurons. Whole-cell proteomic analysis was also performed in neurons. Our results revealed alteration in proteins related to mitochondrial function, cell cycle control, among others. We also performed energy pathway analysis and reactive oxygen species (ROS) analysis of NSCs, which revealed alterations in mitochondrial oxygen consumption and a tendency of higher levels of intracellular ROS in schizophrenia-derived cells.