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OBJECTIVE: To determine the significance of immunological markers in patients with obstructive sleep apnea (OSA) and comorbid pathology. MATERIAL AND METHODS: Sixty-five patients were examined. Two groups of patients were distinguished: the main group with moderate and severe OSA and the control group without OSA. The subjects underwent anthropometry, polysomnography, assessment of cognitive and emotional disorders. Glial fibrillar acidic protein (GFAP), antibodies against NR1-NR2 subunits of NMDA receptors (AT to GRIN2A) and the acetylcholine receptor (AT to AChR), and brain-derived neurotrophic factor (BDNF) were studied by enzyme immunoassay. RESULTS: In patients with OSA, indicators of markers: GFAP (p=0.017), BDNF (p=0.006), antibodies to AChR (p=0.002), as well as chronic cerebral ischemia (p=0.000), depression on the HADS (p=0.004) and the Beck scale (p=0.000), drowsiness on the Epworth scale (p=0.001), asthenia on the visual analogue scale (p=0.000) and the MFI 20 (p=0.013) were higher than in the control group. A relationship was established in the main group between the identified subjective disorders on the Mini-Mental State Examination scale (MMSE) and BDNF (r=0.302, p=0.014) and the average score on the MMSE and BDNF (r=-0.266, p=0.032). CONCLUSION: The results demonstrate the relationship of neurospecific proteins with cognitive impairment in patients with OSA. The neuromarker GFAP in patients with sleep apnea has shown itself to be a predictor of decreased neurogenesis, and BDNF as a representative marker of neuroplasticity. Large values of AT to AChR in patients with OSA may indicate possible neuromuscular transmission disorders. Along with drowsiness and asthenia, patients with OSA have changes in the emotional background, mainly due to depression. The severity of depression and the severity of asthenia increase with increasing severity of apnea and are probably associated with low levels of saturation, which in turn leads to dysregulation of the prefrontal cortex, hippocampus and amygdala.

Asunto(s)

Biomarcadores , Factor Neurotrófico Derivado del Encéfalo , Apnea Obstructiva del Sueño , Humanos , Apnea Obstructiva del Sueño/inmunología , Apnea Obstructiva del Sueño/sangre , Apnea Obstructiva del Sueño/complicaciones , Masculino , Factor Neurotrófico Derivado del Encéfalo/sangre , Persona de Mediana Edad , Femenino , Biomarcadores/sangre , Proteína Ácida Fibrilar de la Glía/sangre , Adulto , Polisomnografía , Comorbilidad , Receptores de N-Metil-D-Aspartato/inmunología , Depresión/sangre , Depresión/epidemiología , Depresión/etiología , Astenia , Anciano

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Bovine herpesvirus 1 (BoHV-1), a significant pathogen in the alpha-herpesvirus subfamily, primarily infects cattle and causes the upper respiratory disease known as infectious bovine rhinotracheitis (IBR). In silico studies evaluated the BoHV-1 D protein to be non-allergenic, non-toxic, and highly antigenic, highlighting its potential as an antigen for vaccine development. Therefore, this study aimed to evaluate the efficacy of a subunit vaccine using the ectodomain of glycoprotein D (gD34-380) as an antigen. The truncated gD was successfully cloned and expressed in both Escherichia coli (E. coli, termed EgD) and baculovirus (termed BgD) systems, with expected molecular weights of 65 kDa and 50 kDa, respectively. For the vaccine formulation, the gD proteins were used either alone or in combination with in-house inactivated BoHV-1. Vaccination of mice and bovines showed that baculovirus-expressed gD34-380 accelerated the antibody response. Moreover, the BgD-vaccinated group also showed significantly higher neutralizing antibody levels against BoHV-1 than the control group (p<0.0001). In conclusion, our study found that BgD from BoHV-1 can increase the immune response and enhance vaccine efficacy.

RESUMEN

Enhancing the efficacy of subunit vaccines relies significantly on the utilization of potent adjuvants, particularly those capable of triggering multiple immune pathways. To achieve synergistic immune augmentation by Toll-like receptor 4 agonist (TLR4a) and nucleotide-binding oligomerization-domain-containing protein 2 agonist (NOD2a), in this work, we conjugated RC529 (TLR4a) and MDP (NOD2a) to give RC529-MDP, and evaluated its adjuvanticity for OVA antigen. Compared to the unconjugated RC529+MDP, RC529-MDP remarkably enhanced innate immune responses with 6.8-fold increase in IL-6 cytokine, and promoted the maturation of antigen-presenting cells (APCs), possibly because of the conjugation of multiple agonists ensuring their delivery to the same cell and activation of various signaling pathways within that cell. Furthermore, RC529-MDP improved OVA-specific antibody response, T cells response and the memory T cells ratio relative to the unconjugated mixture. Therefore, covalently conjugating TLR4 agonist and NOD2 agonist was an effective strategy to enhance immune responses, providing the potential to design and develop more effective vaccines.

Asunto(s)

Acetilmuramil-Alanil-Isoglutamina , Adyuvantes Inmunológicos , Proteína Adaptadora de Señalización NOD2 , Receptor Toll-Like 4 , Proteína Adaptadora de Señalización NOD2/agonistas , Proteína Adaptadora de Señalización NOD2/metabolismo , Receptor Toll-Like 4/agonistas , Animales , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/química , Acetilmuramil-Alanil-Isoglutamina/farmacología , Acetilmuramil-Alanil-Isoglutamina/química , Ratones , Ovalbúmina/inmunología , Humanos , Ratones Endogámicos C57BL , Estructura Molecular , Relación Dosis-Respuesta a Droga , Relación Estructura-Actividad

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Background: Inhibin is a member of the transforming growth factor family that influences reproduction in animals. Objective: The purpose of this study was to obtain nanobodies from the phage antibody library constructed by us that can specifically bind to inhibin α-subunit. Methods: In this study, camels were immunized with Kazakh sheep inhibin-α protein that expressed in BL21 E. coli, and the camel VHH nanobody phage display library was prepared using nested PCR. The nanobodies specifically binding to inhibin α-subunit in the library were screened by three rounds of immunoaffinity screening and phage enzyme-linked immunosorbent assay (phage ELISA). The functions of the selected nanobodies were identified using molecular simulation docking, ELISA affinity test, and sheep immunity test. Results: A nanobody display library was successfully constructed with a capacity of 1.05 × 1012 CFU, and four inhibin-α-subunit-specific nanobodies with an overall similarity of 69.34 % were screened from the library, namely, Nb-4, Nb-15, Nb-26, and Nb-57. The results of molecular simulation docking revealed that four types of nanobodies were complexed with inhibin-α protein mainly through hydrophobic bonds. Immunity tests revealed that the nanobody Nb-4 could effectively inhibit sheep inhibin A/B and could significantly improve the FSH level in sheep. Conclusion: Four inhibin α-subunit-specific nanobodies with biological functions were successfully screened. To the best of our knowledge, this is a new reproductive immunomodulatory pathway of inhibin α-subunit, which may change the secretion of FSH in the ovary, thus changing the estrous cycle of organisms.

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Three fish blood flukes (Aporocotylidae Odhner, 1912) infect mullets (Mugiliformes: Mugilidae): Cardicola mugilis Yamaguti, 1970 and Plethorchis acanthus Martin, 1975 infect striped mullet, Mugil cephalus Linnaeus, 1758 in the Central Pacific Ocean (Hawaiian Islands) and Brisbane River (Australia), respectively; Cardicola brasiliensis Knoff & Amato, 1992 infects Lebranche mullet, Mugil liza Valenciennes, 1836 from the Southwestern Atlantic Ocean (Brazil). White mullets were cast-netted from the mouth of Deer River, a coastal saltmarsh of Mobile Bay, in the north-central Gulf of Mexico and examined for blood fluke infections. Specimens of Mugilitrema labowskiae Warren & Bullard n. gen., n. sp. were found infecting the endocardial surface and inter-trabecular spaces of the atrium, ventricle, and bulbous arteriosus. The new genus and species differ from all other aporocotylids by having the combination of two post-caecal testes, a uterus with straight ascending and descending portions, and a common genital pore. The 28S analysis recovered the new species and P.acanthus as sister taxa and Aporocotylidae as monophyletic. Carditis associated with intense infections comprised endocardial hyperplasia, resulting in a thickened cardiac endothelium. Probable dead or deteriorating eggs in the myocardium were encapsulated by granulomas composed of epithelioid histiocytes. Live eggs infected the afferent artery of gill filaments and were associated with varied hyperplasia of the overlying epithelium and haemorrhaging from the afferent artery in high-intensity infections. The new species is the first aporocotylid infecting a mullet from the northwestern Atlantic Ocean and only the second description of demonstrable endocarditis attributed to an adult fish blood fluke infection.

Asunto(s)

Enfermedades de los Peces , Filogenia , Smegmamorpha , Trematodos , Infecciones por Trematodos , Animales , Trematodos/clasificación , Trematodos/anatomía & histología , Trematodos/aislamiento & purificación , Enfermedades de los Peces/parasitología , Golfo de México , Infecciones por Trematodos/veterinaria , Infecciones por Trematodos/parasitología , Smegmamorpha/parasitología , Bahías , Blanco

RESUMEN

Orf virus (ORFV) is an acute contact, epitheliotropic, zoonotic, and double-stranded DNA virus that causes significant economic losses in the livestock industry. The objective of this study is to design an immunoinformatics-based multi-epitope subunit vaccine against ORFV. Various immunodominant cytotoxic T lymphocytes (CTL), helper T lymphocytes (HTL), and B-cell epitopes from the B2L, F1L, and 080 protein of ORFV were selected and linked by short connectors to construct a multi-epitope subunit vaccine. Immunogenicity was enhanced by adding an adjuvant ß-defensin to the N-terminal of the vaccine using the EAAAK linker. The vaccine exhibited a significant degree of antigenicity and solubility, without allergenicity or toxicity. The 3D formation of the vaccine was subsequently anticipated, improved, and verified. The optimized model exhibited a lower Z-score of -4.33, indicating higher quality. Molecular docking results demonstrated that the vaccine strongly binds to TLR2 and TLR4. Molecular dynamics results indicated that the docked vaccine-TLR complexes were stable. Immune simulation analyses further confirmed that the vaccine can induce a marked increase in IgG and IgM antibody titers, and elevated levels of IFN-γ and IL-2. Finally, the optimized DNA sequence of the vaccine was cloned into the vector pET28a (+) for high expression in the E.coli expression system. Overall, the designed multi-epitope subunit vaccine is highly stable and can induce robust humoral and cellular immunity, making it a promising vaccine candidate against ORFV.

Asunto(s)

Epítopos de Linfocito B , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Virus del Orf , Vacunas de Subunidad , Vacunas Virales , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Vacunas de Subunidad/química , Animales , Virus del Orf/inmunología , Virus del Orf/genética , Vacunas Virales/inmunología , Vacunas Virales/química , Vacunas Virales/genética , Ratones , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/química , Ectima Contagioso/prevención & control , Ectima Contagioso/inmunología , Ectima Contagioso/virología , Ratones Endogámicos BALB C , Femenino , Linfocitos T Citotóxicos/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología

RESUMEN

The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate-generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH-A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers-to-tetramer assembly inhibit human LDH-A (hLDH-A). However, to identify these peptides, tetrameric hLDH-A was transiently exposed to strongly acidic conditions inducing its dissociation into monomers, which were tested as a target for peptides at low pH. Nevertheless, the availability of native monomeric hLDH-A would allow performing similar screenings under physiological conditions. Here we report on the unprecedented isolation of recombinant monomeric hLDH-A at neutral pH, and on its use to identify peptides inhibiting the assembly of the tetrameric enzyme. Remarkably, the GQNGISDL octapeptide, mimicking the 296-303 portion of hLDH-A C-terminal region, was observed to effectively inhibit the target enzyme. Moreover, by dissecting the action of this octapeptide, the cGQND cyclic tetrapeptide was found to act as the parental compound. Furthermore, we performed assays using MCF7 and BxPC3 cultured cells, exclusively expressing hLDH-A and hLDH-B, respectively. By means of these assays we detected a selective action of linear and cyclic GQND tetrapeptides, inhibiting lactate secretion in MCF7 cells only. Overall, our observations suggest that peptides mimicking the C-terminal region of hLDH-A effectively interfere with protein-protein interactions responsible for the assembly of the tetrameric enzyme.

Asunto(s)

L-Lactato Deshidrogenasa , Ácido Láctico , Multimerización de Proteína , Humanos , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/química , Ácido Láctico/metabolismo , Ácido Láctico/química , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Concentración de Iones de Hidrógeno , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/genética , Oligopéptidos/farmacología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Línea Celular Tumoral

RESUMEN

INTRODUCTION: Gemcitabine (GEM) is the first-line drug for pancreatic ductal adenocarcinoma (PDAC), but drug resistance severely restricts its chemotherapeutic efficacy. Laminin subunit γ2 (LAMC2) plays a crucial role in extracellular matrix formation in the development of GEM-resistance. However, the biological function of LAMC2 in GEM resistance and its molecular mechanisms are still unclear. 20(S)-Ginsenoside Rh2 (Rh2), one of the principal active components isolated from Ginseng Radix et Rhizoma, possesses strong anti-tumor effects. However, the effects of Rh2 on overcoming GEM resistance and its action mechanisms remain to be elucidated. OBJECTIVES: This study aimed to determine the efficacy of Rh2 on overcoming GEM resistance and to explore its underlying molecular mechanisms. METHODS: Clinical study, Western blotting, publicly available databasesand bioinformatic analyses were performed to investigate the protein expression of LAMC2 in the GEM-resistant PDAC patients and the acquired GEM-resistant PDAC cells. Then, the effects of Rh2 on overcoming the GEM resistance in PDAC were evaluated both in vitro and in vivo. Stable silencing or overexpression of LAMC2 in the GEM-resistant PDAC cells were established for validating the role of LAMC2 on Rh2 overcoming the GEM resistance in PDAC. RESULTS: The protein expression of LAMC2 was markedly increased in the GEM-resistant PDAC patient biopsies compared to the sensitive cases. The protein expression of LAMC2 was significantly higher in the acquired GEM-resistant PDAC cells than that in their parental cells. Rh2 enhanced the chemosensitivity of GEM in the GEM-resistant PDAC cells, and inhibited the tumor growth of Miapaca-2-GR cell-bearing mice and Krastm4TyjTrp53tm1BrnTg (Pdx1-cre/Esr1*) #Dam/J (KPC) mice. Rh2 effectively reversed the GEM resistance in Miapaca-2-GR and Capan-2-GR cells by inhibiting LAMC2 expression through regulating the ubiquitin-proteasome pathway. Knockdown of LAMC2 enhanced the chemosensitivity of GEM and the effects of Rh2 on overcoming the GEM resistance in PDAC cells and the orthotopic PDAC mouse model. Conversely, LAMC2 overexpression aggravated the chemoresistance of GEM and abolished the effects of Rh2 on overcoming GEM resistance via modulating ATP-binding cassette (ABC) transporters leading to the active GEM efflux. CONCLUSIONS: LAMC2 plays an important role in the GEM resistance in PDAC, and Rh2 is a potential adjuvant for overcoming the chemoresistance of GEM in PDAC.

RESUMEN

(1): Atopic dermatitis and psoriasis vulgaris are chronic, inflammatory diseases. Clinical presentation usually leads to a proper diagnosis, but sometimes neither clinical examination nor histopathological evaluation can be conclusive. Therefore, we aimed to build up a novel diagnostic tool and check it for accuracy. The main objective of our work was to differentiate between healthy skin (C), atopic dermatitis (AD) and psoriasis vulgaris (PV) biopsies on the base of involucrin (IVL) and human ß-defensin-2 (hBD-2) concentrations and their mRNA, as well as mRNA for TPP2 and PSMB8. (2): ELISA for IVL and hBD-2 proteins and Real-time PCR for the relative expression of mRNA for: IVL (IVL mRNA), hBD-2 (hBD-2 mRNA), PSMB8 (PSMB8 mRNA) and TPP2 (TPP2 mRNA), isolated from skin biopsies taken from AD and PV patients and healthy volunteers were performed. (3): hBD-2 mRNA and PSMB8 mRNA correlated with some parameters of clinical assessment of inflammatory disease severity. hBD-2 mRNA expression, exclusively, was sufficient to distinguish inflammatory skin biopsies from the healthy ones. (4): hBD-2 mRNA and PSMB8 mRNA analysis were the most valuable parameters in differentiating AD and PV biopsies.

Asunto(s)

Dermatitis Atópica , Psoriasis , ARN Mensajero , Piel , beta-Defensinas , Humanos , Psoriasis/genética , Psoriasis/metabolismo , Psoriasis/patología , Psoriasis/diagnóstico , beta-Defensinas/genética , beta-Defensinas/metabolismo , Dermatitis Atópica/genética , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Dermatitis Atópica/diagnóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Biopsia , Femenino , Masculino , Piel/metabolismo , Piel/patología , Adulto , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Diagnóstico Diferencial , Adulto Joven , Adolescente

RESUMEN

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent chronic liver condition worldwide, demanding further investigation into its pathogenesis. Circular RNAs (circRNAs) are emerging as pivotal regulators in MASLD processes, yet their pathological implications in MASLD remain poorly understood. This study focused on elucidating the role of circular RNA ribonucleotide reductase subunit M2 (circRRM2) in MASLD progression. In this study, we used both in vitro and in vivo MASLD models using long-chain-free fatty acid (FFA)-treated hepatocytes and high-fat diet (HFD)-induced MASLD in mice, respectively. We determined the expression patterns of circRRM2, microRNA-142-5p (miR-142-5p), and neuregulin 1 (NRG1) in livers of MASLD-afflicted mice and MASLD hepatocytes by RT-qPCR. Dual-luciferase reporter assays verified the binding relationships among circRRM2, miR-142-5p, and NRG1. We conducted further analyses of their roles in MASLD hepatocytes and modulated circRRM2, miR-142-5p, and NRG1 expression in vitro by transfection. Our findings were validated in vivo. The results demonstrated reduced levels of circRRM2 and NRG1, along with elevated miR-142-5p expression in MASLD livers and hepatocytes. Overexpression of circRRM2 downregulated lipogenesis-related genes and decreased triglycerides accumulation in livers of MASLD mice. MiR-142-5p, which interacts with circRRM2, effectively counteracted the effects of circRRM2 in MASLD hepatocytes. Furthermore, NRG1 was identified as a miR-142-5p target, and its overexpression mitigated the regulatory impact of miR-142-5p on MASLD hepatocytes. In conclusion, circRRM2, via its role as a miR-142-5p sponge, upregulating NRG1, possibly influenced triglycerides accumulation in both in vitro and in vivo MASLD models.NEW & NOTEWORTHY CircRRM2 expression was downregulated in free fatty acid (FFA)-challenged hepatocytes and high-fat diet (HFD) fed mice. Overexpressed circular RNA ribonucleotide reductase subunit M2 (circRRM2) attenuated metabolic dysfunction-associated steatotic liver disease (MASLD) development by suppressing FFA-induced triglycerides accumulation. CircRRM2 targeted microRNA-142-5p (miR-142-5p), which served as an upstream inhibitor of neuregulin 1 (NRG1) and collaboratively regulated MASLD progression.

Asunto(s)

Dieta Alta en Grasa , Hepatocitos , MicroARNs , Neurregulina-1 , ARN Circular , Animales , MicroARNs/metabolismo , MicroARNs/genética , Ratones , Hepatocitos/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Masculino , Neurregulina-1/genética , Neurregulina-1/metabolismo , Ratones Endogámicos C57BL , Hígado Graso/metabolismo , Hígado Graso/genética , Humanos , Hígado/metabolismo , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Ribonucleósido Difosfato Reductasa

RESUMEN

In skin aging, it has been hypothesized that aging fibroblasts accumulate within the epidermal basal layer, dermis, and subcutaneous fat, causing abnormal tissue remodeling and extracellular matrix dysfunction, thereby inducing an aging-related secretory phenotype (SASP). A new treatment for skin aging involves the specific elimination of senescent skin cells, especially fibroblasts within the dermis and keratinocytes in the basal layer. This requires the identification of specific protein markers of senescent cells, such as ribonucleoside-diphosphate reductase subunit M2 B (RRM2B), which is upregulated in various malignancies in response to DNA stress damage. However, the behavior and role of RRM2B in skin aging remain unclear. Therefore, we examined whether RRM2B functions as a senescence marker using a human dermal fibroblast model of aging. In a model of cellular senescence induced by replicative aging and exposure to ionizing radiation or UVB, RRM2B was upregulated at the gene and protein levels. This was correlated with decreased uptake of the senescence-associated ß-galactosidase activity and proliferation marker bromodeoxyuridine. RRM2B upregulation was concurrent with the increased expression of SASP factor genes. Furthermore, using fluorescence flow cytometry, RRM2B-positive cells were recovered more frequently in the aging cell population. In aging human skin, RRM2B was also found to be more abundant in the dermis and epidermal basal layer than other proteins. Therefore, RRM2B may serve as a clinical marker to identify senescent skin cells.

RESUMEN

Gallein, a small molecule related to fluorescein, is established as an inhibitor of Gßγ subunits to inhibit G protein (Gs) signaling. This agent is providing a potential therapeutic strategy to ameliorate organ dysfunctions especially involved in inflammation, however; the effects on bone metabolism have not yet been clarified. Prostaglandins (PGs) play important roles as autacoids including osteoblasts, and d-type prostanoid (DP) receptor, a member of G protein-coupled receptor specific to PGD2, is expressed on osteoblasts. We previously reported that prostaglandin D2 (PGD2) induces the syntheses of osteoprotegerin (OPG) and interleukin-6 (IL-6), essential factors in bone remodelling process, and p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/p42 MAPK are involved in the signal transduction of PGD2 in osteoblast-like MC3T3-E1 cells. Thus, we investigated in this study that the effect and the underlying mechanism of gallein, an inhibitor Gßɤ subunits, on the syntheses of OPG and IL-6 induced by PGD2 in these cells. The cultured cells were treated with gallein or fluorescein, a structurally related compound inactive to Gßɤ subunits, and subsequently stimulated with PGD2. Not fluorescein but gallein amplified the PGD2-stimulated releases of OPG and IL-6. Gallein enhanced the PGD2-upregulated mRNA expression levels of OPG and IL-6. Regarding the signaling mechanism, gallein did not affect the PGD2-induced phosphorylation of p38 MAPK, JNK, or p42 MAPK. In conclusion, gallein upregulates the PGD2-stimulated syntheses of OPG and IL-6 by the specific effect to inhibit Gßγ subunits in osteoblasts, but the effect is not exerted at the upstream of p38 MAPK, JNK, or p44/p42 MAPK activation.

RESUMEN

METHODS: Single-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1ß. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action. RESULTS: CFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1ß, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3). CONCLUSION: The study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs.

Asunto(s)

Hidrogeles , Degeneración del Disco Intervertebral , Núcleo Pulposo , Péptidos , Regeneración , Animales , Hidrogeles/química , Núcleo Pulposo/metabolismo , Ratones , Degeneración del Disco Intervertebral/terapia , Regeneración/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Disco Intervertebral , Humanos , Proliferación Celular/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Movimiento Celular/efectos de los fármacos

RESUMEN

BACKGROUND: Fusarium head blight (FHB) significantly impacts wheat yield and quality. Understanding the intricate interaction mechanisms between Fusarium graminearum (the main pathogen of FHB) and wheat is crucial for developing effective strategies to manage and this disease. Our previous studies had shown that the absence of the cell wall mannoprotein FgCWM1, located at the outermost layer of the cell wall, led to a decrease in the pathogenicity of F. graminearum and induced the accumulation of salicylic acid (SA) in wheat. Hence, we propose that FgCWM1 may play a role in interacting between F. graminearum and wheat, as its physical location facilitates interaction effects. RESULTS: In this study, we have identified that the C-terminal region of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9) could interact with FgCWM1 through the yeast two-hybrid assay. The interaction was further confirmed through the combination of Co-IP and BiFC analyses. Consistently, the results of subcellular localization indicated that TaNDUFA9 was localized in the cytoplasm adjacent to the cell membrane and chloroplasts. The protein was also detected to be associated with mitochondria and positively regulated complex I activity. The loss-of-function mutant of TaNDUFA9 exhibited a delay in flowering, decreased seed setting rate, and reduced pollen fertility. However, it exhibited elevated levels of SA and increased resistance to FHB caused by F. graminearum infection. Meanwhile, inoculation with the FgCWM1 deletion mutant strain led to increased synthesis of SA in wheat. CONCLUSIONS: These findings suggest that TaNDUFA9 inhibits SA synthesis and FHB resistance in wheat. FgCWM1 enhances this inhibition by interacting with the C-terminal region of TaNDUFA9, ultimately facilitating F. graminearum infection in wheat. This study provides new insights into the interaction mechanism between F. graminearum and wheat. TaNDUFA9 could serve as a target gene for enhancing wheat resistance to FHB.

Asunto(s)

Resistencia a la Enfermedad , Fusarium , Enfermedades de las Plantas , Proteínas de Plantas , Ácido Salicílico , Triticum , Triticum/microbiología , Triticum/genética , Triticum/metabolismo , Enfermedades de las Plantas/microbiología , Fusarium/fisiología , Resistencia a la Enfermedad/genética , Ácido Salicílico/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo

RESUMEN

Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups, leading to their systematics remaining unresolved. Phylogenetic relationships within the hypotrich family Strongylidiidae have been ambiguous due to discordance between the morphological and genetic data. In this study, a new strongylidiid genus Heterouroleptus is established, mainly based on the novel mode of origin of the ventral cirral rows: left ventral cirral row (LVR) originates from frontal-ventral-transverse cirral anlagen (FVTA) III (anterior portion), IV (middle portion), and V (rear portion); right ventral cirral row comes from the entire FVTA VI. A new species, Heterouroleptus weishanensis gen. nov., sp. nov., is investigated along with the morphometric and molecular data from a population of Strongylidium wuhanense. Eight new sequences and nuclear gene markers (single-gene and multi-gene) are provided to analyze the phylogenetic relationships of strongylidiids, with the COI gene utilized to uncover further genetic information at species level and below. The results reveal that: (1) Strongylidiidae is monophyletic and has a close relationship with Dorsomarginalia; (2) Heterouroleptus gen. nov. forms a clade that is sister to all the other strongylidiids; (3) Hemiamphisiella Foissner, 1988 and Pseudouroleptus Hemberger, 1985 should not be synonyms, and both genera should be subdivided due to their variable morphological characteristics; (4) LVR originating from three anlagen is a plesiomorphy of Strongylidiidae. The discovery of the origin of the LVR not only contributes to the establishment of the genus Heterouroleptus, but also helps to improve the diagnosis of the family Strongylidiidae. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00243-z.

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Introduction: Gluten quality is one of the most important traits of the common wheat (Triticum aestivum L.). In Chinese wheat production, Yannong series cultivars/derivative lines possess unique characteristics and play an important role in both yield and quality contribution. Methods: To dissect their genetic basis of the gluten quality, in this study, allelic variations of high-molecular-weight glutenin subunit (HMW-GS) and low-molecular-weight glutenin subunit (LMW-GS) in 30 Yannong series wheat cultivars/derivative lines and three check cultivars were evaluated using the allele-specific molecular markers, and six crucial quality indexes were also further measured and analyzed. Results: The results demonstrated that the frequencies of HMW-GSs By8, Dx5+Dy10 and Dx5+Dy10+Dy12 in these 30 genotypes and three check cultivars accounted for 87.9%, 24.2% and 9.1%, respectively. For the allelic variations of LMW-GSs, Glu-A3a, Glu-A3b, Glu-A3c, Glu-A3f, and Glu-A3g were identified in 18, 9, 13, 11, and 2 genotypes, respectively; Glu-B3d, Glu-B3g and Glu-B3f were identified in 13, 23 and 4 genotypes, respectively. Notably, Yannong 999, containing By8 + Dx5 + Dy10, and Jinan 17 containing By8 + Dy12 both meet the national standard for high-quality wheat and belong to the category of first-class high-quality strong gluten wheat. Discussion: These findings can provide reference for wheat quality improvement and popularization in the production.

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Parkinson's disease (PD) involves alpha-synuclein accumulation according to Braak's pattern, with diverse clinical progressions that complicate diagnosis and treatment. We aimed to correlate Braak's pattern with rapid progressive PD to identify blood-based biomarkers and therapeutic targets exploiting brown algae-derived bioactives for potential treatment. We implemented a systematic workflow of transcriptomic profiling, co-expression networks, cluster profiling, transcriptional regulator identification, molecular docking, quantum calculations, and dynamic simulations. The transcriptomic analyses exhibited highly expressed genes at each Braak's stage and in rapidly progressive PD. Co-expression networks for Braak's stages were built, and the top five clusters from each stage displayed significant overlap with differentially expressed genes in rapidly progressive PD, indicating shared biomarkers between the blood and the PD brain. Further investigation showed, NF-kappa-B p105 as the master transcriptional regulator of these biomarkers. Molecular docking screened phlorethopentafuhalol-A from brown algae, exhibiting a superior inhibitory effect with p105 (- 7.51 kcal/mol) by outperforming PD drugs and anti-inflammatory compounds (- 5.73 to - 4.38 kcal/mol). Quantum mechanics and molecular mechanics (QM/MM) calculations and dynamic simulations have confirmed the interactive stability of phlorethopentafuhalol-A with p105. Overall, our combined computational study shows that phlorethopentafuhalol-A derived from brown algae, may have healing properties that could help treat PD.

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We have investigated the hippocampal connectivity of the marmoset presubiculum (PreS) and reported that major connections of PreS in the rat were conserved in the marmoset. Moreover, our results indicated the presence of several additional projections that were almost absent in the rat brain, but abundant in the marmoset, such as direct projections from CA1 to PreS. However, little is known about the connectivity between the frontal brain regions and PreS or hippocampal formation. Therefore, we investigated the distribution of cells of the origins and terminals of the presubicular and hippocampal projections in the marmoset frontal brain regions using the retrograde and anterograde tracer cholera toxin B subunit. In cases of tracer injections into all layers of PreS, many neurons and terminals were labeled in the claustrum-endopiriform (Cl-En) complex almost entirely along the rostrocaudal axis. Even in cases where the injection site involved the superficial (not deep) layers of PreS, labeled neurons and terminals were distributed over a wide rostrocaudal range of the Cl-En complex, but their number and density were significantly lower than the whole-layer injection cases. In cases where the injection site was confined to the hippocampal formation, labeled cells and terminals were localized at a restricted portion of the Cl-En complex. Here, we demonstrate for what we believe to be the first time the strong, reciprocal connections of the Cl-En complex with PreS and projections from the Cl-En complex to the hippocampal regions (CA1 and the subiculum) in the marmoset. Our findings indicate that the Cl-En complex may exert a strong influence on the cortical and subcortical outputs from PreS and, in turn, the entire memory circuitry in the marmoset brain.

Asunto(s)

Callithrix , Claustro , Hipocampo , Vías Nerviosas , Animales , Callithrix/anatomía & histología , Hipocampo/anatomía & histología , Hipocampo/citología , Vías Nerviosas/anatomía & histología , Vías Nerviosas/citología , Masculino , Claustro/anatomía & histología , Claustro/fisiología , Femenino , Neuronas/citología , Toxina del Cólera/metabolismo

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Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time-consuming. Using a reverse workflow for specimen sorting and identification leveraging high-throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology-based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity.

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Chaperonins/Heat shock protein 60 are ubiquitous multimeric protein complexes that assist in the folding of partially and/or misfolded proteins using metabolic energy into their native stage. The eukaryotic group II chaperonin, also referred as T-complex protein-1 ring complex (TRiC)/T-complex protein-1 (TCP1)/chaperonin containing T-complex protein (CCT), contains 8-9 paralogous subunits, arranged in each of the two rings of hetero-oligomeric complex. In Leishmania, till date, only one subunit, LdTCP1γ, has been well studied. Here, we report the molecular, structural, and functional characterization of TCP1δ subunit of Leishmania donovani (LdTCP1δ), the causative agent of Indian kala-azar. LdTCP1δ gene exhibited only 27.9% identity with LdTCP1γ and clustered in a separate branch in the phylogenic tree of LdTCP1 subunits. The purified recombinant protein formed a high molecular weight complex (0.75 MDa), arranged into 16-mer assembly, and performed in vitro chaperonin activity as assayed by ATP-dependent luciferase folding. LdTCP1δ exhibits 1.8-fold upregulated expression in metabolically active, rapidly dividing log phase promastigotes. Over-expression of LdTCP1δ in promastigotes results in increased infectivity and rate of multiplication of intracellular amastigotes. The study thus establishes the existence of an individual functionally active homo-oligomeric complex of LdTCP1δ chaperonin with its role in parasite infectivity and multiplication.