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Understanding the responses of olive trees to drought stress is crucial for improving cultivation and developing drought-tolerant varieties. Water transport and storage within the plant is a key factor in drought-tolerance strategies. Water management can be based on a variety of factors such as stomatal control, osmoprotectant molecules, proteins and wood properties. The aim of the study was to evaluate the water management strategy under drought stress from an anatomical and biochemical point of view in three young Italian olive cultivars (Giarraffa, Leccino and Maurino) previously distinguished for their physiological and metabolomic responses. For each cultivar, 15 individuals in pots were exposed or not to 28 days of water withholding. Every 7 days, the content of sugars (including mannitol), proline, aquaporins, osmotins, and dehydrins, in leaves and stems, as well as the chemical and anatomical characteristics of the wood of the three cultivars, were analyzed. 'Giarraffa' reduced glucose levels and increased mannitol production, while 'Leccino' accumulated more proline. Both 'Leccino' and 'Maurino' increased sucrose and aquaporin levels, possibly due to their ability to remove embolisms. 'Maurino' and 'Leccino' accumulated more dehydrins and osmotins. While neither genotype nor stress affected wood chemistry, 'Maurino' had a higher vessel-to-xylem area ratio and a larger hydraulic diameter, which allows it to maintain a high transpiration rate but may make it more susceptible to cavitation. The results emphasized the need for an integrated approach, highlighting the importance of the relative timing and sequence of each parameter analyzed, allowing, overall, to define a "strategy" rather than a "response" to drought of each cultivar. [ABSTRACT FROM AUTHOR]

Introduction Cigarette smoking greatly promotes the progression and poor prognosis of colorectal cancer (CRC) patients, with the molecular mechanism still not fully clear. Methods In this study, CRC cells were exposed to tobacco-specific nitrosamine 4‑(methylnitrosamino)‑1‑(3‑pyridyl)-1‑butanone (NNK), and the differentially expressed smoking-related genes were identified based on both NNK-induced CRC cells and a total of 763 CRC tissues from The Cancer Genome Atlas cohort. Cox regression analysis, receiver operating characteristic curve and Kaplan–Meier plot were used to establish the risk score model for CRC prognosis. Moreover, quantitative real-time–PCR, western blotting, colony formation, migration, and invasion assays were performed to verify the core differentially expressed smoking-related gene and its molecular function in NNK-induced CRC progression. Results Results indicated NNK significantly enhanced CRC cell proliferation, migration and invasion. Moreover, a four-gene signature containing AKR1B10, CALB2, PLAC1, and GNA15 was established as a CRC prognosis marker. Among these four genes, AKR1B10 was further validated as the core gene, and its expression was significantly inhibited after NNK exposure in CRC cells. Results of gene enrichment analysis and western blotting suggested AKR1B10 might reduce the malignant progression of NNK-induced CRC cells by inhibiting the Wnt signaling pathway by promoting E-Cadherin expression and inhibiting the expression of N-Cadherin, β-Catenin, Vimentin, and Snail. Conclusions In conclusion, new four smoking-related genes can be jointly used as prognostic markers for CRC. AKR1B10 served as a tumor suppressor, and can be used as a potential target to inhibit NNK-induced CRC malignant progression by regulating the Wnt signaling pathway. Implications This study demonstrates that tobacco-derived NNK dependence would promote the malignant progression of colorectal cancer by regulating the expressions of the AKR1B10/Wnt signaling pathway. A novel four-gene signature is established for the prognosis prediction of smoking CRC patients. These findings have important translational implications given the continued use of tobacco and the difficulty in smoking cessation worldwide, which can be applied to alleviate the adverse effects induced by tobacco dependence on colorectal cancer patients. [ABSTRACT FROM AUTHOR]

Background: Defects in PPP1R12A can lead to genitourinary and/or brain malformation syndrome (GUBS). GUBS is primarily characterized by neurological or genitourinary system abnormalities, but a few reported cases are associated with neonatal seizures. Here, we report a case of a female newborn with neonatal seizures caused by a novel variant in PPP1R12A, aiming to enhance the clinical and variant data of genetic factors related to epilepsy in early life. Methods: Whole-exome and Sanger sequencing were used for familial variant assessment, and bioinformatics was employed to annotate the variant. A structural model of the mutant protein was simulated using molecular dynamics (MD), and the free binding energy between PPP1R12A and PPP1CB was analyzed. A mutant plasmid was constructed, and mutant protein expression was analyzed using western blotting (WB), and the interaction between the mutant and PPP1CB proteins using co-immunoprecipitation (Co-IP) experiments. Results: The patient experienced tonic-clonic seizures on the second day after birth. Genetic testing revealed a heterozygous variant in PPP1R12A, NM_002480.3:c.2533 C > T (p.Arg845Ter). Both parents had the wild-type gene. MD suggested that loss of the C-terminal structure in the mutant protein altered its structural stability and increased the binding energy with PPP1CB, indicating unstable protein–protein interactions. On WB, a low-molecular-weight band was observed, indicating that the protein was truncated. Co-IP indicated that the mutant protein no longer interacted with PPP1CB, indicating an effect on the structural stability of the myosin phase complex. Conclusion: The PPP1R12A c.2533 C > T variant may explain the neonatal seizures in the present case. The findings of this study expand the spectrum of PPP1R12A variants and highlight the potential significance of truncated proteins in the pathogenesis of GUBS. [ABSTRACT FROM AUTHOR]

BACKGROUND: The temporal and spatial expression of fibroblast growth factor receptors 1 and 2 remains a controversial issue during kidney development, so the relationship between them and kidney development remains unclear. OBJECTIVE: To observe the dynamic expression of fibroblast growth factor receptors 1 and 2 during kidney development of mice, and to investigate the relationship between them and kidney development. METHODS: The kidneys of fetal mice [embryotic days (E) 12, 14, 16, and 18] and neonatal mice [neonatal days (N) 1, 3, 7, 14, 24, and 40] were selected to examine the temporal and spatial expression of fibroblast growth factor receptors 1 and 2 by immunohistochemistry method in kidney tissues, and quantitative analysis was performed using western blot assay. RESULTS AND CONCLUSION: (1) Immunohistochemistry showed that fibroblast growth factor receptor 1 was mainly localized in metanephric tissue surrounding the tip of the ureteral bud at E12. Subsequently, fibroblast growth factor receptor 1 was expressed in immature renal corpuscles at various stages, some distal convoluted tubules and capillary loops. The positive site was mainly concentrated in the generative region. Fibroblast growth factor receptor 2 was initially expressed in both ureteral buds and metanephric tissue. Fibroblast growth factor receptor 2 was localized in immature renal corpuscles, distal tubules, collecting ducts and thin segments of medullary loops with kidney development. However, the expression of renal corpuscles was weak. (2) Stereology and western blot assay showed that the expression of fibroblast growth factor receptor 1 was high before birth and gradually decreased after birth, while the expression was very low after N7 day. The expression level of fibroblast growth factor receptor 2 increased gradually with the kidney development and tended to be stable after N7 day. (3) The results exhibit that fibroblast growth factor receptors 1 and 2 are expressed spatially and temporally during kidney development. It is speculated that fibroblast growth factor receptors 1 and 2 may influence nephron development and maturation, and fibroblast growth factor receptor 2 is critical during the formation of ureteral buds and morphology. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the degradation pattern of cardiac troponin I in rats in vivo, and to determine whether the pattern was dependent on the cause of death, for the purpose of estimating the postmortem interval. The rats were categorized into three distinct groups depending on the factors leading to their demise: the control group, the group experiencing acebutolol-induced cardiotoxicity, and the group affected by asphyxia. The analysis encompassed the isolation and segregation of the protein, subsequently employing Western blotting as a means of visualizing the results. The results revealed a distinct degradation pattern of cTnI into smaller fragments over time, indicating that cardiac troponin I can serve as a reliable marker for estimating the postmortem interval. Furthermore, noteworthy variations were noted in the degradation pattern of cardiac troponin I among the different causes of death, which suggests that this method can also be used to determine whether cardiac failure was the cause of death or not. [ABSTRACT FROM AUTHOR]

Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season. • MLT concentration in goats' seminal plasma does not show interseasonal variation. • Expression of MLT receptors in goat spermatozoa shows variation throughout the year. • Goat buck sperm freezability is associated with the expression of MLT receptors. [ABSTRACT FROM AUTHOR]

Purpose: Glioma-associated oncogene homolog-1 (GLI1) is amplified in human glioblastoma, and there is growing evidence suggesting its significant role in tumor development and metastasis. Our aim was to investigate the role of the GLI-1 gene in the progression of colorectal cancer (CRC) and its correlation with various clinicopathological features. Additionally, we examined the impact of the GLI-1 gene and other factors on the prognosis of CRC. Methods: We analyzed a total of 98 confirmed CRC cases and adjacent normal tissue controls. Patients suspected of having colon cancer underwent a colonoscopy and targeted biopsy, while those with rectal cancer underwent CT scans and MRI. GLI1 expression was detected using real-time PCR assay, Western blotting, and immunohistochemistry. Results: The GLI1 gene was observed to be overexpressed in tumor tissues at both the protein and mRNA levels (p < 0.05). In addition, GLI1 overexpression was significantly associated with various factors such as tumor invasion (T3/T4), presence of lymph nodes, lymph node metastasis (LNM), stage (III/IV), tumor site (colon), tumor size (≥ 3 cm), localization (nucleocytoplasmic), strong staining intensity and recurrence (p < 0.05). The results of survival analysis showed that the patients with overexpression of GLI1 had a significantly lower DFS rate which was 21 months compared to those with normal expression who had 31 months (p < 0.05). Moreover, individuals with early onset disease (15 months) were more likely to have cytoplasmic localization of the GLI1 gene as opposed to nucleo-cytoplasmic localization of GLI1 which presented late-onset disease(23 months) (p < 0.05). Finally, Stage and PNI (p < 0.05) were found to independently affect outcomes of CRC according to Cox regression analysis. Conclusion: High expression of GLI-1 in CRC is associated with adverse pathology and poor prognosis for patients. The correlation between cytoplasmic localization of GLI-1 and reduced disease-free survival holds potential for guiding prognosis and treatment. Further research is needed to develop strategies targeting GLI-1 for improved outcomes. [ABSTRACT FROM AUTHOR]

El presente estudio se planteó determinar el rendimiento de antígenos de Leishmania braziliensis y Leishmania peruviana en la detección de LTA, fue desarrollado a partir de muestras de suero obtenidas entre 2013 - 2016. Los antígenos solubles y de excreción/secreción obtenidos fueron transferidos a membrana de nitrocelulosa mediante un ensayo de inmunotransferencia. Se realizó la evaluación frente a sueros confirmados para LTA, a un nivel de confianza al 95%, logrando determinar que, el antígeno soluble de Leishmania braziliensis presenta una sensibilidad del 87,7%, especificidad del 100% y área bajo la curva de 0,95; mientras que, Leishmania peruviana se encontró valores de 92,3%, 95,7% y 0,94 respectivamente. De acuerdo a los resultados, recomendamos realizar la caracterización y análisis de las regiones inmunogénicas reportadas a fin de continuar con el desarrollo de proteínas recombinantes y sintéticas, orientadas a mejorar la eficiencia del diagnóstico serológico de la enfermedad.

Abstract Background Defects in PPP1R12A can lead to genitourinary and/or brain malformation syndrome (GUBS). GUBS is primarily characterized by neurological or genitourinary system abnormalities, but a few reported cases are associated with neonatal seizures. Here, we report a case of a female newborn with neonatal seizures caused by a novel variant in PPP1R12A, aiming to enhance the clinical and variant data of genetic factors related to epilepsy in early life. Methods Whole-exome and Sanger sequencing were used for familial variant assessment, and bioinformatics was employed to annotate the variant. A structural model of the mutant protein was simulated using molecular dynamics (MD), and the free binding energy between PPP1R12A and PPP1CB was analyzed. A mutant plasmid was constructed, and mutant protein expression was analyzed using western blotting (WB), and the interaction between the mutant and PPP1CB proteins using co-immunoprecipitation (Co-IP) experiments. Results The patient experienced tonic-clonic seizures on the second day after birth. Genetic testing revealed a heterozygous variant in PPP1R12A, NM_002480.3:c.2533 C > T (p.Arg845Ter). Both parents had the wild-type gene. MD suggested that loss of the C-terminal structure in the mutant protein altered its structural stability and increased the binding energy with PPP1CB, indicating unstable protein–protein interactions. On WB, a low-molecular-weight band was observed, indicating that the protein was truncated. Co-IP indicated that the mutant protein no longer interacted with PPP1CB, indicating an effect on the structural stability of the myosin phase complex. Conclusion The PPP1R12A c.2533 C > T variant may explain the neonatal seizures in the present case. The findings of this study expand the spectrum of PPP1R12A variants and highlight the potential significance of truncated proteins in the pathogenesis of GUBS.

Phlebotomus perniciosus is a major vector of Leishmania infantum in the Mediterranean. While the seroprevalence of leishmaniosis in Spanish dogs and cats has been studied, data on the exposure of cats to P. perniciosus bites under natural conditions without repellents is limited. Stray cats could serve as sentinels for L. infantum and P. perniciosus exposure. This study analyzed sera from 204 apparently healthy stray cats, collected from January 2021 to January 2022, for antibodies against P. perniciosus saliva and L. infantum parasites. Anti-sand fly antibodies were detected in 40.69% of cats using an ELISA with the recombinant salivary protein SP03B of P. perniciosus. Seroprevalence of L. infantum infection was 23.52% by Western blot and 27.41% by ELISA, with an overall seroprevalence of 40.69% (95% CI 34.18–47.54%). This is the first assessment of antibody response to P. perniciosus saliva and L. infantum in naturally exposed stray cats in Spain. Further research is needed to examine the salivary antigens recognized by cats and to explore the relationship between P. perniciosus exposure and L. infantum infection severity in cats.

ABSTRACTPrion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt–Jakob disease (sCJD), Gerstmann–Sträussler–Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8–9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

The use of human monoclonal antibodies in diagnostics and analytical studies is determined by the rapid development of technologies and approaches in the field of immunology and clinical laboratory diagnostics. Cytokines, as biologically active substances, play a key role in the regulation of immunity, inflammation and other physiological processes. The use of monoclonal antibodies to cytokines provides an opportunity to accurately determine their concentration and study the interaction with other molecules in clinical studies. The development of new diagnostic methods and analytical studies using monoclonal antibodies opened up prospects for increasing the accuracy and sensitivity of the determination of various diseases. This is especially important in the context of medicine, where accurate diagnosis becomes fundamental for choosing the optimal method. Aim. The purpose of this work was to analyze the possibilities and advantages of using monoclonal antibodies to human cytokines in diagnostics and analytical studies, in particular their use in immunological biosensors, for monitoring the immunological status of patients after organ transplantation and diagnosing oncological diseases. Methods. A comprehensive review of literature was conducted, encompassing multiple databases including Medline (PubMed), Web of Science, Scopus, Google Scholar and other relevant sources up to the year 2024. Keywords employed in the search included biosensors, cell markers, monoclonal antibodies, leukemia and Western blotting, transplantation. Results. The paper investigated the potential and advantages of using human monoclonal antibodies to cytokines in medical diagnostics and analytical research. Their application in biosensors for precise determination of cytokine concentrations, immunophenotyping of blood cells and tissues, diagnosing tumors of various histogenesis, treating leukemias and lymphomas, determining immunological status and composition of the immune system cells were analyzed. The methodology of comprehensive immunological examination after organ transplantation and treatment, including аcquired immunodeficiency syndrome (AIDS), was also considered. Conclusions. Monoclonal antibodies, effective in biosensors, are used in various fields such as biological research, clinical diagnostics, and environmental monitoring. Their use has limitations, such as cost, complexity of acquisition, and potential loss of signal sensitivity. Immunophenotyping allows the identification of blood cells based on their markers, which is important for hematological diagnostics. Immunohistochemical tissue studies use monoclonal antibodies to verify tumors and detect infectious diseases. Diagnosis of leukemias and lymphomas using monoclonal antibodies helps to determine tumor type and optimal treatment. Western blotting is used to determine proteins in tissues or blood, including confirming diseases such as Lyme disease or Human immunodeficiency virus (HIV infection. Flow cytometry, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) are also used in immunological laboratories for disease diagnosis and immune status assessment.

Melanin naturally exists in organisms and is synthetized by tyrosinase (TYR); however, its over-production may lead to aberrant pigmentation and skin conditions. Loquat (Eriobotrya japonica (Thunb.) Lindl.) flowers contain a variety of bioactive compounds, while studies on their suppressive capabilities against melanin synthesis are limited. Loquat flower isolate product (LFP) was obtained by ethanol extraction and resin purification, and its inhibitory efficiency against TYR activity was investigated by enzyme kinetics and multiple spectroscopy analyses. In addition, the impact of LFP on melanin synthesis-related proteins' expression in mouse melanoma B16 cells was analyzed using Western blotting. HPLC-MS/MS analysis indicated that LFP was composed of 137 compounds, of which 12 compounds, including flavonoids (quercetin, isorhamnoin, p-coumaric acid, etc.) and cinnamic acid and its derivatives, as well as benzene and its derivatives, might have TYR inhibitory activities. LFP inhibited TYR activity in a concentration-dependent manner with its IC50 value being 2.8 mg/mL. The inhibition was an anti-competitive one through altering the enzyme's conformation rather than chelating copper ions at the active center. LFP reduced the expression of TYR, tyrosinase-related protein (TRP) 1, and TRP2 in melanoma B16 cells, hence inhibiting the synthesis of melanin. The research suggested that LFP had the potential to reduce the risks of hyperpigmentation caused by tyrosinase and provided a foundation for the utilization of loquat flower as a natural resource in the development of beauty and aging-related functional products. [ABSTRACT FROM AUTHOR]

Delivering nucleic acid therapeutics across cell membranes is a significant challenge. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) show promise for siRNA delivery. To improve siRNA delivery and silence a model STAT3 gene, we hypothesized that oleyl acylation to CPPs, specifically (WRH)n, would enhance STAT3 silencing efficiency in breast and ovarian cancer cells. Using Fmoc/tBu solid-phase peptide chemistry, we synthesized, purified, and characterized the oleyl-conjugated (WRH)n (n = 1–4) peptides. The peptide/siRNA complexes were non-cytotoxic at N/P 40 (~20 μM) against MDA-MB-231, MCF-7, SK-OV-3, and HEK-293 cells after 72 h incubation. All peptide/siRNA complexes showed serum stability at N/P ≥ 40. The synthesized conjugates, with a diameter of <100 nm, formed nano-complexes with siRNA and exhibited a stable range of zeta potential values (13–18 mV at N/P = 40). Confocal microscopy and flow cytometry analysis provided qualitative and quantitative evidence of a successful cellular internalization of siRNA. The peptides oleyl-(WRH)3 and oleyl-(WRH)4 showed ~60% and ~75% cellular uptake of siRNA, respectively, in both MDA-MB-231 and SK-OV-3 cells. Western blot analysis of oleyl-(WRH)4 demonstrated effective silencing of the STAT-3 gene, with ~75% silencing in MDA-MB-231 cells and ~45% in SK-OV-3 cells. [ABSTRACT FROM AUTHOR]

In the pathological process of Alzheimer's disease, neuronal cell death is closely related to the accumulation of reactive oxygen species. Our previous studies have found that oxidative stress can activate microtubule affinity-regulating kinases, resulting in elevated phosphorylation levels of tau protein specifically at the Ser262 residue in N1E-115 cells that have been subjected to exposure to hydrogen peroxide. This process may be one of the pathogenic mechanisms of Alzheimer's disease. Vitamin E is a fat-soluble, naturally occurring antioxidant that plays a crucial role in biological systems. This study aimed to examine the probable processes that contribute to the inhibiting effect on the abnormal phosphorylation of tau protein and the neuroprotective activity of a particular type of vitamin E, α-tocotrienol. The experimental analysis revealed that α-tocotrienol showed significant neuroprotective effects in the N1E-115 cell line. Our data further suggest that one of the mechanisms underlying the neuroprotective effects of α-tocotrienol may be through the inhibition of microtubule affinity-regulated kinase activation, which significantly reduces the oxidative stress-induced aberrant elevation of p-Tau (Ser262) levels. These results indicate that α-tocotrienol may represent an intriguing strategy for treating or preventing Alzheimer's disease. [ABSTRACT FROM AUTHOR]

One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10−8 M and 7.3 × 10−9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies. [ABSTRACT FROM AUTHOR]

Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial–mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation. [ABSTRACT FROM AUTHOR]

Purpose: The contribution of androgen receptors (AR) on bladder cancer has been demonstrated in pre-clinical studies, however in clinical studies, only the canonical AR (AR-FL) protein was measured by immunohistochemistry and conflicting results were obtained. To get better insight into the alterations of AR signalling, we used western blotting (WB) method and simultaneously measured both mRNA and protein levels of AR-FL and AR-V7. Methods: 23 naive non-muscle invasive bladder cancer patients and 12 healthy individuals were included. AR-FL protein, AR-FL mRNA, AR-V7 protein and AR-V7 mRNA levels were quantitatively measured by WB and qRT-PCR. Results: While AR-FL protein and AR-V7 mRNA were significantly higher in bladder cancer, AR-FL mRNA and AR-V7 protein were lower. AR-V7 mRNA level was higher in patients with tumour size over 3 cm and AR-FL protein was higher in single tumours (p < 0,005). The small sampling size and the inclusion of only male participants were the main limitations. Conclusions: The increase of AR-FL protein in bladder cancer supports the contribution of the AR pathway in bladder cancer. The presence of high AR-FL protein despite low mRNA levels may be due to a disruption in post-transcriptional regulatory mechanisms. AR-V7 was demonstrated for the first time in bladder tissue and found significantly different in bladder cancer tissues. Our study reached new and valuable findings and will shed light on the studies that aim to clarify the role of the AR pathway in bladder cancer. [ABSTRACT FROM AUTHOR]

This study aimed to determine the performance of Leishmania braziliensis and Leishmania peruviana antigens in the detection of ATL by using serum samples obtained between 2013 - 2016. The obtained soluble and excretion/secretion antigens were transferred to membrane nitrocellulose by immunoblot assay. The evaluation was carried out against sera confirmed for ATL, at a confidence level of 95%, determining that the soluble antigen of Leishmania braziliensis had a sensitivity of 87.7%, specificity of 100% and area under the curve of 0.95; on the other hand, Leishmania peruviana showed values of 92.3%, 95.7% and 0.94, respectively. According to the results, we recommend that the reported immunogenic regions should be characterized and analyzed in order to continue with the development of recombinant and synthetic proteins, aimed at improving the efficiency of the serological diagnosis of the disease. [ABSTRACT FROM AUTHOR]

Cannabis is an annual herb of the genus Cannabis, with a history of medical use going back thousands of years. However, its abuse causes many side-effects, including confusion of consciousness, alienation, and mental disorders such as schizophrenia and depression. Research conducted on rodents suggests that there are two types of cannabinoid receptors—cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R). CB1R is found mostly in the central nervous system, particularly in the prefrontal cortex (PFC), and alterations in its expression in the PFC have been strongly linked to mental disorders. Within the layers of the PFC, Brodmann area 46 is associated with the processing of complex cognitive information. However, it remains unclear whether CB1R is expressed in the PFC 46 area of non-human primate. In this work, we applied western blotting along with immunofluorescent histochemical staining to investigate the distribution pattern of CB1R in the PFC of nonhuman primate, Our findings reveal that CB1R is highly expressed in the monkey PFC, especially in area 46. Furthermore, CB1R exhibits a layered distribution pattern within area 46 of the PFC, with the inner granular layer displaying the highest expression levels. Additionally, CB1R+PV+ cells are widely distributed in lay II-VI of area 46, with layer IV showing notable prevalence. In conclusion, CB1R is distributed in the PV interneurons in area 46 of the prefrontal cortex, particularly in layer IV, suggesting that cannabis may modulate PFC activities via regulating interneuron in the PFC. And cannabis-induced side effects may be caused by abnormal expression of CB1R. [ABSTRACT FROM AUTHOR]

The utility of serum glial fibrillary acidic protein (GFAP) in acute ischemic stroke (AIS) has been extensively studied in recent years. Here, we aimed to assess its potential role as a cargo protein of extracellular vesicles (EVs) secreted by astrocytes (ADEVs) in response to brain ischemia. Plasma samples from eighteen AIS patients at 24 h (D1), 7 days (D7), and one month (M1) post-symptoms onset, and nine age, sex, and cardiovascular risk factor-matched healthy controls were obtained to isolate EVs using the Exoquick ULTRA EV kit. Subsets of presumed ADEVs were identified further by the expression of the glutamate aspartate transporter (GLAST) as a specific marker of astrocytes with the Basic Exo-Flow Capture kit. Western blotting has tested the presence of GFAP in ADEV cargo. Post-stroke ADEV GFAP levels were elevated at D1 and D7 but not M1 compared to controls (p = 0.007, p = 0.019, and p = 0.344, respectively). Significant differences were highlighted in ADEV GFAP content at the three time points studied (n = 12, p = 0.027) and between D1 and M1 (z = 2.65, p = 0.023). A positive correlation was observed between the modified Rankin Scale (mRS) at D7 and ADEV GFAP at D1 (r = 0.58, p = 0.010) and D7 (r = 0.57, p = 0.013), respectively. ADEV GFAP may dynamically reflect changes during the first month post-ischemia. Profiling ADEVs from peripheral blood could provide a new way to assess the central nervous system pathology. [ABSTRACT FROM AUTHOR]

Abstract Background Limb Girdle Muscular Dystrophy R1 (LGMDR1) is an autosomal recessive neuromuscular disease caused by mutations in the calpain-3 (CAPN3) gene. As clinical and pathological features may overlap with other types of LGMD, therefore definite molecular diagnosis is required to understand the progression of this debilitating disease. This study aims to identify novel variants of CAPN3 gene in LGMDR1 patients. Results Thirty-four patients with clinical and histopathological features suggestive of LGMD were studied. The muscle biopsy samples were evaluated using Enzyme histochemistry, Immunohistochemistry, followed by Western Blotting and Sanger sequencing. Out of 34 LGMD cases, 13 patients were diagnosed as LGMDR1 by immunoblot analysis, demonstrating reduced or absent calpain-3 protein as compared to controls. Variants of CAPN3 gene were also found and pathogenicity was predicted using in-silico prediction tools. The CAPN3 gene variants found in this study, included, two missense variants [CAPN3: c.1189T > C, CAPN3: c.2338G > C], one insertion-deletion [c.1688delinsTC], one splice site variant [c.2051-1G > T], and one nonsense variant [c.1939G > T; p.Glu647Ter]. Conclusions We confirmed 6 patients as LGMDR1 (with CAPN3 variants) from our cohort and calpain-3 protein expression was significantly reduced by immunoblot analysis as compared to control. Besides the previously known variants, our study found two novel variants in CAPN3 gene by Sanger sequencing-based approach indicating that genetic variants in LGMDR1 patients may help to understand the etiology of the disease and future prognostication.

Plumage color, a pivotal attribute delineating diverse Muscovy duck strains, assumes considerable significance within the field of Muscovy duck breeding research. This study extends the existing research by delving into the hereditary aspects of genes associated with plumage coloration in Muscovy ducks. The principal objective is to discern marker genes conducive to targeted breeding strategies based on plumage color, thereby furnishing indispensable technical foundations for the development of novel Muscovy duck varieties. Our investigation focused on scrutinizing the impact of MYOT and MB genes on the genetic expression of plumage color at both the RNA and protein levels in Muscovy ducks. The results elucidate that black Muscovy ducks manifest markedly elevated mRNA and protein expression levels of MYOT and MB genes in comparison to their white counterparts, indicating that both genes may play a constructive regulatory role in the context of plumage coloration in Muscovy ducks. The outcomes of this study delineate a discernible correlation between MYOT and MB genes and the plumage coloration in Muscovy ducks. Employing gene expression analysis, we successfully identified candidate genes that may be intricately linked to the determination of plumage color in these ducks.

Leukocyte migration is an essential function of innate and adaptive immune responses. Chemokines and their receptors control the migration system. The abundance of chemokines is controlled by atypical chemokine receptors (ACKRs), chemokine receptor-like molecules that do not couple to the G protein signaling pathways. Among them, ACKR4 regulates dendritic cell migration by controlling the ligands and is involved in tumor development in mouse models. Because no anti-mouse ACKR4 (mACKR4) monoclonal antibody (mAb) for flow cytometry has been reported, this study aimed to develop a novel mAb for mACKR4. Among the established anti-mACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab-2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant (KD) values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/mACKR4 were determined as 6.0 × 10−9 M, 1.3 × 10−8 M, and 1.7 × 10−9 M, respectively. Furthermore, A4Mab-1 and A4Mab-2 could detect mACKR4 by western blotting. These results indicated that A4Mab-1, A4Mab-2, and A4Mab-3 help to detect mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.

A 68‐year‐old woman presented with difficulty finding words and writing characters. Neurological examination led to clinical diagnosis at onset of the logopenic variant of primary progressive aphasia accompanied with ideomotor apraxia, visuospatial agnosia on the right, and Gerstmann syndrome. Bradykinesia and rigidity on the right with shuffling gait developed after one year. Treatment with L‐dopa had no effect. The patient was diagnosed with corticobasal syndrome (CBS). Brain magnetic resonance imaging revealed diffuse cortical atrophy dominantly on the left, especially in the temporal, parietal, and occipital lobes. Positron emission tomography did not reveal any significant accumulation of amyloid β or tau protein. She died five years later. Neuropathological examination revealed diffuse cortical atrophy with severe neuronal loss and fibrous gliosis in the cortex. Neuronal cytoplasmic inclusions, short dystrophic neurites, and, most notably, neuronal intranuclear inclusions, all immunoreactive for phosphorylated TDP‐43, were observed. Western blotting revealed a full length and fragments of phosphorylated TDP‐43 at 45 and 23 kDa, respectively, confirming the pathological diagnosis of type A FTLD‐TDP. Whole exome sequencing revealed a pathogenic mutation in GRN (c.87dupC). FTLD‐TDP should be included in the differential diagnosis of CBS. [ABSTRACT FROM AUTHOR]

Background: Obsessive-Compulsive Disorder (OCD) is a type of anxiety disorder that is marked by intrusive and distressing thoughts, as well as repetitive behaviors. Trout fish (Oncorhynchus mykiss) is a functional food that might have potential therapeutic effects on many neurological disorders including OCD. Objective: This study aims to explore the effects of Al-Assi River trout fish meal, a dietary source of tryptophan, on obsessive-compulsive disorder (OCD) symptoms and related molecular pathways in BALB/c mice. Methods: OCD mice were divided into five groups: one control group without any treatment, one group treated with fluoxetine (a selective serotonin reuptake inhibitor), and three groups fed with different doses of trout fish meal (0, 7.5, and 15 g/kg body weight). The mice were subjected to various behavioral tests, such as the marble test, tail suspension test, sucrose preference test, and forced swim test, to evaluate OCD and depressive-like behaviors. Moreover, the expression and protein levels of genes involved in the serotonergic and GABAergic systems were measured. Results: The results indicated that trout fish meal had dose-dependent effects on OCD-like behaviors, revealing exacerbation at lower doses and improvement at higher doses. For instance, in the marble test, OCD mice fed with 7.5 g of trout fish/kg body weight buried more marbles than those fed with 15 g/kg of trout fish (4.5 vs 3.33 out of 6, p>0.05). In the tail suspension test, the immobility time of OCD mice treated with fluoxetine was numerically lower than that of the untreated OCD mice (63.6 vs 87.3 seconds, p>0.05). Furthermore, normal mice had different baseline gene expression profiles than OCD mice. Normal mice had the highest fold increase of Gabra gene expression (3.75) compared to the untreated OCD group, followed by groups treated with 7.5 and 15 g of trout fish/kg body weight (2.02 and 1.44, respectively). Conclusions: This study suggests that dietary interventions rich in tryptophan, such as trout fish meal, may have modulatory effects on OCD symptoms and molecular mechanisms in mice. However, the optimal dosing and individual variability need to be considered. More research is required to clarify the underlying mechanisms and to evaluate the potential efficacy of trout fish meal in treating OCD in humans. [ABSTRACT FROM AUTHOR]

Programmed cell death‐ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single‐chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof‐of‐concept data for the effect of scFv using PDL1‐expressing cells was lacking. In this study, we conducted two kinds of cell‐based immunoassays, western blotting and enzyme‐linked immunosorbent assay, using anti‐PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell‐based immunoassays to detect PDL1. [ABSTRACT FROM AUTHOR]

Idiopathic normal pressure hydrocephalus in elderly people is considered a form of glymphopathy caused by malfunction of the waste clearance pathway, called the glymphatic system. Tau is a representative waste material similar to amyloid-β. During neurodegeneration, lipocalin-type prostaglandin D synthase (L-PGDS), a major cerebrospinal fluid (CSF) protein, is reported to act as a chaperone that prevents the neurotoxic aggregation of amyloid-β. L-PGDS is also a CSF biomarker in idiopathic normal pressure hydrocephalus and significantly correlates with tau concentration, age, and age-related brain white matter changes detected by magnetic resonance imaging. To investigate this glymphopathy, we aimed to analyze white matter changes and contributing factors in vivo and their interactions ex vivo. Cerebrospinal tap tests were performed in 60 patients referred for symptomatic ventriculomegaly. Patients were evaluated using an idiopathic normal pressure hydrocephalus grading scale, mini-mental state examination, frontal assessment battery, and timed up-and-go test. The typical morphological features of high convexity tightness and ventriculomegaly were measured using the callosal angle and Evans index, and parenchymal white matter properties were evaluated with diffusion tensor imaging followed by tract-based spatial statistics. Levels of CSF biomarkers, including tau, amyloid-β, and L-PGDS, were determined by ELISA, and their interaction, and localization were determined using immunoprecipitation and immunohistochemical analyses. Tract-based spatial statistics for fractional anisotropy revealed clusters that positively correlated with mini-mental state examination, frontal assessment battery, and callosal angle, and clusters that negatively correlated with age, disease duration, idiopathic normal pressure hydrocephalus grading scale, Evans index, and L-PGDS. Other parameters also indicated clusters that correlated with symptoms, microstructural white matter changes, and L-PGDS. Tau co-precipitated with L-PGDS, and colocalization was confirmed in postmortem specimens of neurodegenerative disease obtained from the human Brain Bank. Our study supports the diagnostic value of L-PGDS as a surrogate marker for white matter integrity in idiopathic normal pressure hydrocephalus. These results increase our understanding of the molecular players in the glymphatic system. Moreover, this study indicates the potential utility of enhancing endogenous protective factors to maintain brain homeostasis. [ABSTRACT FROM AUTHOR]

Background: Limb Girdle Muscular Dystrophy R1 (LGMDR1) is an autosomal recessive neuromuscular disease caused by mutations in the calpain-3 (CAPN3) gene. As clinical and pathological features may overlap with other types of LGMD, therefore definite molecular diagnosis is required to understand the progression of this debilitating disease. This study aims to identify novel variants of CAPN3 gene in LGMDR1 patients. Results: Thirty-four patients with clinical and histopathological features suggestive of LGMD were studied. The muscle biopsy samples were evaluated using Enzyme histochemistry, Immunohistochemistry, followed by Western Blotting and Sanger sequencing. Out of 34 LGMD cases, 13 patients were diagnosed as LGMDR1 by immunoblot analysis, demonstrating reduced or absent calpain-3 protein as compared to controls. Variants of CAPN3 gene were also found and pathogenicity was predicted using in-silico prediction tools. The CAPN3 gene variants found in this study, included, two missense variants [CAPN3: c.1189T > C, CAPN3: c.2338G > C], one insertion-deletion [c.1688delinsTC], one splice site variant [c.2051-1G > T], and one nonsense variant [c.1939G > T; p.Glu647Ter]. Conclusions: We confirmed 6 patients as LGMDR1 (with CAPN3 variants) from our cohort and calpain-3 protein expression was significantly reduced by immunoblot analysis as compared to control. Besides the previously known variants, our study found two novel variants in CAPN3 gene by Sanger sequencing-based approach indicating that genetic variants in LGMDR1 patients may help to understand the etiology of the disease and future prognostication. [ABSTRACT FROM AUTHOR]

The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation. Because it is essential to understand the disease of giant panda for conservation, histopathological analyses of tissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors, which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognized N-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) in flow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 × 10−8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells. [ABSTRACT FROM AUTHOR]

Background Enhancing graft fat survival remains a paramount challenge in autologous fat transplantation surgeries. Dedifferentiated fat cells (DFATs) and adipose-derived stem cells (ASCs) represent 2 pivotal cells with potential to improve fat graft survival rates. Objectives In this study we aimed to compare the effectiveness of DFATs and ASCs in promoting fat graft survival, emphasizing their adipogenic and angiogenic capabilities. Methods Both in vitro and in vivo experiments were conducted. In vitro assessments compared adipogenesis, angiogenesis, osteogenesis, chondrogenesis, cell migration abilities, and surface markers. For in vivo evaluation, a cell-assisted lipotransfer animal model was employed to gauge graft volume retention and histological morphology. Analysis techniques included hematoxylin and eosin staining, Western blotting, and real-time polymerase chain reaction. Results In vitro findings suggested a slight superiority of DFATs in adipogenesis and angiogenesis compared to ASCs. In vivo tests demonstrated both cell types surpassed the control in terms of graft volume retention, with the DFATs group marginally outperforming in retention rates and the ASC group presenting a slightly enhanced graft tissue structure. Conclusions Our study underscores the distinct advantages of DFATs and ASCs in bolstering fat graft survival, offering potentially novel insights for plastic surgeons aiming to elevate fat graft survival rates. [ABSTRACT FROM AUTHOR]

Hong Kong oysters (Crassostrea hongkongensis) are an important marine bivalve with nutritional and commercial value. The expanded off-bottom farming scale in recent years makes the oysters more susceptible to exposure to abiotic stresses, such as salinity stress, an important environmental factor that has been proven to have significant effects on oyster growth and development. However, the molecular mechanism is still unclear. Cyclin E is an important protein in the process of cell cycle regulation that is indispensable for propelling G1/S phase transition in a dose-dependent manner. In order to investigate whether the salinity stress affects cyclin E expression in oysters, the cDNA sequence of C. hongkongensis cyclin E (Ch-CCNE) was isolated from a gill cDNA library, and the 2.8 kbp length cDNA fragment contained a complete open reading frame (ORF) encoding 440 amino acid residues. Ch-CCNE mRNA was highly expressed in the gonad and low in the adductor mussel, mantle, gill, labial palp, and digestive gland. The recombinant CCNE protein was expressed and purified in a pET32a(+)-CCNE/Escherichia coli BL21(DE3) system via IPTG induction and was used for generating mice anti-Ch-CCNE antiserums. Western blot analysis showed that the CCNE protein in the gill was maintained at low expression levels under either hypo- (5 ppt) or hyper- (35 ppt) salinity, and could be produced at high levels under appropriate salinity during a 10-day exposure period. The immuno-localization indicated that the Ch-CCNE protein was distributed in the nucleus. These results suggested that either hypo- or hyper-salinity stress could inhibit the CCNE expression of Hong Kong oysters and their negative impact on cell division and proliferation. [ABSTRACT FROM AUTHOR]

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research. [ABSTRACT FROM AUTHOR]

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Introduction: Serological assays are alternative laboratory tools for the diagnosis of parasitic infections. The aim of this work was to evaluate the performance of the indirect fluorescent antibody test (IFAT) and Western blotting (WB) for the detection of IgG anti-Giardia antibodies in human sera. Methodology: Sera from individuals infected with Giardia duodenalis, other parasites or non-parasitized were selected for serological assays. Ninety-seven sera were tested by IFAT at 1:20 and 1:40 dilutions and of these, 40 samples were also analyzed by WB. Results: The sensitivity and specificity of the IFAT was 97% and 46.9% at 1:20 sera dilution, and 39.4% and 59.4% at 1:40 sera dilution. The low molecular weight polypeptides fractions of 25 kDa, 27–31 kDa and 45–55 kDa were the most frequently identified by the sera of individuals infected with G. duodenalis, along with low cross-reactivity, presenting an individual sensitivity of 42.8%, 50.0% and 57.1%, and specificity of 83.3%, 83.3% and 91.7%, respectively. The highest overall sensitivity of WB (85.7%) was based on the immunoreactivity of sera with at least one of those proteins. The concordance between the detection of G. duodenalis in feces by microscopy and the WB results was considered substantial (Kappa = 0.61). Conclusion: Constant exposure to Giardia infection throughout a lifetime can maintain high levels of specific antibodies in serum, even without active infection. Moreover, proteins found in intestinal amoebas may hinder the serological diagnosis of giardiasis in endemic areas due to cross-reactivity, which can be partially solved using Giardia low molecular weight proteins. [ABSTRACT FROM AUTHOR]

This article has tried to provide an overview of the most sophisticated microfluidic biosensors for identifying nucleic acids and proteins at the site of treatment. Microfluidics, which also automates sample preparation and reduces processing time and reagent consumption, enables the analysis of small sample quantities. Microfluidics and biosensor technologies collaborate to provide diagnostics at the point of care with high throughput analysis, portability, and disposability. The high sensitivity and selectivity requirements, false response errors, and integration with other essential modules are some of the challenges posed by this merger. The broad categories of protein-based and DNA-based biosensor technology are covered in this review. Also, recent advancements in coupling the biosensors to microfluidics, the main challenges and potential solutions in deploying microfluidic biosensors for point-of-care diagnostics, and the most recent developments in these areas have been discussed.

Diagnosis of light chain amyloidosis (AL) requires demonstration of amyloid deposits in a tissue biopsy followed by appropriate typing. Previous studies demonstrated increased dimerization of monoclonal serum free light chains (FLCs) as a pathological feature of AL. To further examine the pathogenicity of FLC, we aimed at testing amino acid sequence homology between circulating and deposited light chains (LCs). Matched tissue biopsy and serum of 10 AL patients were subjected to tissue proteomic amyloid typing and nephelometric FLC assay, respectively. Serum FLC monomers (M) and dimers (D) were analyzed by Western blotting (WB) and mass spectrometry (MS). WB of serum FLCs showed predominance of either κ or λ type, in agreement with the nephelometric assay data. Abnormal FLC M–D patterns typical of AL amyloidosis were demonstrated in 8 AL-λ patients and in one of two AL-κ patients: increased levels of monoclonal FLC dimers, high D/M ratio values of involved FLCs, and high ratios of involved to uninvolved dimeric FLCs. MS of serum FLC dimers showed predominant constant domain sequences, in concordance with the tissue proteomic amyloid typing. Most importantly, variable domain sequence homology between circulating and deposited LC species was demonstrated, mainly in AL-λ cases. This is the first study to demonstrate homology between circulating FLCs and tissue-deposited LCs in AL-λ amyloidosis. The applied methodology can facilitate studying the pathogenicity of circulating FLC dimers in AL amyloidosis. The study also highlights the potential of FLC monomer and dimer analysis as a non-invasive screening tool for this disease. [ABSTRACT FROM AUTHOR]

To further refine the near-infrared spectroscopy (NIRS)-derived measure of skeletal muscle oxidative capacity in humans, we sought to determine whether the exercise stimulus intensity affected the τ value and/or influenced the magnitude of correlations with in vitro measures of mitochondrial content and in vivo indices of exercise performance. Males (n = 12) and females (n = 12), matched for maximal aerobic fitness per fat-free mass, completed NIRS-derived skeletal muscle oxidative capacity tests for the vastus lateralis following repeated contractions at 40% (`40) and 100% (τ100) of maximum voluntary contraction, underwent a skeletal muscle biopsy of the same muscle, and performed multiple intermittent isometric knee extension tests to task failure to establish critical torque (CT). The value of τ100 (34.4 ± 7.0 s) was greater than τ40 (24.2 ± 6.9 s, P < 0.001), but the values were correlated (r = 0.688; P < 0.001). The values of τ40 (r = -0.692, P < 0.001) and τ100 (r = -0.488, P = 0.016) correlated with myosin heavy chain I percentage and several markers of mitochondrial content, including COX II protein content in whole muscle (s40: r = -0.547, P = 0.006; τ100: r = -0.466, P = 0.022), type I pooled fibers (s40: r = -0.547, P = 0.006; τ100: r = -0.547, P = 0.006), and type II pooled fibers (s40: r = -0.516, P = 0.009; τ100: r = -0.635, P = 0.001). The value of τ40 (r = -0.702, P < 0.001), but not τ100 (r = -0.378, P = 0.083) correlated with critical torque (CT); however, neither value correlated with W0 (τ40: r = 0.071, P = 0.753; τ100: r = 0.054, P = 0.812). Overall, the NIRS method of assessing skeletal muscle oxidative capacity is sensitive to the intensity of skeletal muscle contraction but maintains relationships to whole body fitness, isolated limb critical intensity, and mitochondrial content regardless of intensity. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) is a common malignancy in women of reproductive age. Circular RNAs (circRNAs) are emerging players in OC progression. We investigated the function and mechanism of circular RNA hsa_circ_0027803 (circCDK17) in OC pathogenesis. Real‑time PCR (RT-qPCR) and western blot were utilized for gene and protein expression analysis, respectively. Cell counting kit‑8 (CCK-8), EdU and Transwell assays investigated OC cell proliferation, migration and invasion. The associations between circCDK17, miR-22-3p and CD147 were examined by dual-luciferase reporter and RNA-protein immunoprecipitation (RIP) assays. The in vivo model of OC nude mice was constructed to explore the role of circCDK17. CircCDK17 was increased in OC tissue and cells, and patients with higher expression of circCDK17 had a shorter survival. CircCDK17 downregulation inhibited OC cell proliferation, migration and invasion, and reduced epithelial-mesenchymal transition (EMT)-related markers. In vivo experiments showed that circCDK17 silencing inhibited OC tumor growth and metastasis. CircCDK17 depletion reduced CD147 level via sponging miR-22-3p. MiR-22-3p knockdown overturned effect of circCDK17 depletion on OC cell proliferation, migration and invasion. Meanwhile, overexpressed CD147 restored functions of circCDK17 downregulation on OC development. CircCDK17 is an important molecule that regulates OC pathogenic process through miR-22-3p/CD147. [ABSTRACT FROM AUTHOR]

Objective Gastric cancer (GC) is prevalent worldwide but has a dismal prognosis, and its molecular and pathogenic pathways remain unknown. Kallikrein 11 (KLK11) has a reduced expression in GC and may be a promising biomarker. Method Herein, the function of KLK11 in GC and its regulatory mechanism was studied. Gene sequencing and quantitative reverse transcription-polymerase chain reaction were used to determine the expression of KLK11 in GC and precancerous lesions. Cell function tests and flow cytometry were conducted to determine the proliferative capacity and cell cycle of GC cells, respectively. A luciferase reporter test confirmed the interaction between RNA molecules. The mTOR/4E-BP1 signaling pathway was analyzed using western blotting. Result KLK11 has a suppressed expression in GC samples. KLK11 decreased the proliferative capacity of GC cells, by inhibiting the degree of mTOR/4E-BP1 phosphorylation. In contrast, miR-1304 increased GC cell proliferation by inhibiting KLK11. Moreover, KLK11 was able to limit in vivo GC cell proliferation. Conclusion These findings reveal a promising strategy to prevent and treat GC by targeting the KLK11-mediated mTOR/4E-BP1 cascade. [ABSTRACT FROM AUTHOR]

Cyprinid herpesvirus 3 （CyHV3）, also known as Koi herpesvirus （KHV）, is a highly contagious pathogen that threatens the sustainability of cyprinid aquaculture. The ORF134 gene of CyHV3 encodes an interleukin （IL） 10 like protein. In this study, we constructed a prokaryotic expression plasmid of ORF134 and transformed it into the E. coli Rosetta （DE3） cells for expression of recombinant protein. The recombinant CyHV3-IL10 protein was purified by size exclusion chromatography and used for immunization of mice to generate monoclonal antibodies. The antibodies were then characterized by ELISA, Western blotting and immunofluorescent assay. SDS-PAGE analysis showed that the recombinant CyHV3-IL10 protein was approximately 18 ku in size and was expressed mainly in the form of inclusion bodies. The inclusion bodies were denatured, refolded and subjected to size exclusion chromatography. The recombinant CyHV3-IL10 protein was of high purity. Western blotting and immunofluorescence analyses revealed that the CyHV3-IL10 monoclonal antibodies recognized the recombinant CyHV3-IL10 protein expressed in the E. coli and HEK293 cells and could specifically detect the HEK293 cells expressing the CyHV3-IL10 protein. In summary, the availability of CyHV3-IL10 monoclonal antibodies lays the foundation for the functional study of CyHV3-IL10 and the establishment of CyHV3 diagnostic tools, and provides novel strategies for the prevention and control of the diseases caused by CyHV3. [ABSTRACT FROM AUTHOR]

Cutaneous malignant melanoma is one of the most lethal cutaneous malignancies. Accumulating evidence has demonstrated the potential influence of long non-coding RNAs (lncRNAs) in biological behaviors of melanoma. Herein, we reported a novel lncRNA, lnc-PKNOX1-1 and systematically studied its functions and possible molecular mechanisms in melanoma. Reverse transcription-quantitative PCR assay showed that lnc-PKNOX1-1 was significantly decreased in melanoma cells and tissues. Low lnc-PKNOX1-1 expression was significantly correlated with invasive pathological type and Breslow thickness of melanoma. In vitro and in vivo experiments showed lnc-PKNOX1-1 dramatically inhibited melanoma cell proliferation, migration and invasion. Mechanically, protein microarray analysis suggested that interleukin-8 (IL-8) was negatively regulated by lnc-PKNOX1-1 in melanoma, which was confirmed by western blot and ELISA. Western blot analysis also showed that lnc-PKNOX1-1 could promote p65 phosphorylation at Ser536 in melanoma. Subsequent rescue assays proved IL-8 overexpression could partly reverse the tumor-suppressing function of lnc-PKNOX1-1 overexpression in melanoma cells, indicating that lnc-PKNOX1-1 suppressed the development of melanoma by regulating IL-8. Taken together, our study demonstrated the tumor-suppressing ability of lnc-PKNOX1-1 in melanoma, suggesting its potential as a novel diagnostic biomarker and therapeutic target for melanoma. [ABSTRACT FROM AUTHOR]

Introduction: Oleogum resins extracted from Boswellia sacra (Frankincense) and Commiphora myrrha (Myrrha) have been traditionally used to facilitate wound healing and address skin injuries. Moreover, they have anti-inflammatory, antioxidant, and antimicrobial effects. Therefore, we hypothesized that their combination can be effective in wound healing. In this study, we evaluated the effects of methanol extracts from two oleogum resins, Boswellia sacra (Frankincense) and Commiphora myrrha (Myrrha), as well as their combination on cell migration promotion and wound healing in human dermal fibroblast cells (HDFa). Methods: The methanol extracts of B. sacra (BS) and C. myrrha (CM) and their combination were tested to determine their optimum cytoprotective concentrations using the AlamarBlue assay. The level of reactive oxygen species (ROS) was also evaluated using a DCFDA detector. To assess cell migration promotion and wound healing properties of the extracts, a scratch wound closure assay was performed in HDFa cells and the images were analyzed using ImageJ software. Western blot analysis was employed to detect the activation of fibroblast migrationassociated protein extracellular signal-regulated kinase (ERK). Results: Using the viability assay, the optimum non-cytotoxic concentrations of the extracts (10 and 20 µg/ml) were chosen to evaluate their wound healing effects on HDFa cells. BS, CM and BC at 10 and 20 µg/ml significantly reduced H2 O2 -induced ROS levels compared to the control. In the scratch assay, BS and BC, both at 10 µg/ml, could significantly reduce the average wound width compared to the control. Western blot analysis showed that CM significantly increased the pERK/ERK ratio compared to the control. Conclusion: These findings suggest the beneficial effects of both frankincense and myrrh, as well as their combination, in improving proliferation, migration, and thecwound healing process in HDFa. [ABSTRACT FROM AUTHOR]

Aim: To investigate the effects of Tupichinol E, an alkaloid present in Tupistra chinensis BAKER (Liliaceae), on the metastasis of triple-negative mammary carcinoma in vitro and elucidate the underlying mechanisms. Tupichinol E binding to EGFR stabilizes the protein structure and is a pharmacologically active substance that can be used against cancer due to its potent activity. Methods: The growth of MCF-7 and MDA-MB-231 cells was assessed using the MTT assay. The apoptosis and cell cycle of MCF-7 cells were evaluated with flow cytometry, and the related proteins were examined using Western blotting. Results: Tupichinol E (50–200 μmol/L) inhibits the growth of MCF-7 cells in a dose- and time-dependent manner (the IC50 values are 85.05 ± 1.08 and 50.52 ± 1.06 μmol/L, respectively, at 48 and 72 h). Treatment of MCF-7 cells with Tupichinol E (50–200 μmol/L) dose-dependently induced apoptosis of MCF-7 cells, accompanying the activation of caspase 3. The cells treated with Tupichinol E (100 and 200 μmol/L) significantly increased the percentage of cells in the G2/M phase with a reduction in the expression of cyclin B1. Conclusion: These results demonstrated that Tupichinol E is an effective anti-tumor compound in vitro and has the potential to be developed as a new anticancer drug.

Aims: Currently, the effect of drug treatment for osteoporosis is relatively poor, and the side effects are numerous and serious. Melatonin is a potential drug to improve bone mass in postmenopausal women. Unfortunately, the mechanism by which melatonin improves bone metabolism remains unclear. The aim of this study was to further investigate the potential mechanism of melatonin in the treatment of osteoporosis. Methods: The effects of melatonin on mitochondrial apoptosis protein, bmal1 gene, and related pathway proteins of RAW264.7 (mouse mononuclear macrophage leukaemia cells) were analyzed by western blot. Cell Counting Kit-8 was used to evaluate the effect of melatonin on cell viability. Flow cytometry was used to evaluate the effect of melatonin on the apoptosis of RAW264.7 cells and mitochondrial membrane potential. A reactive oxygen species (ROS) detection kit was used to evaluate the level of ROS in osteoclast precursors. We used bmal1-small interfering RNAs (siRNAs) to downregulate the Bmal1 gene. We established a postmenopausal mouse model and verified the effect of melatonin on the bone mass of postmenopausal osteoporosis in mice via micro-CT. Bmal1 lentiviral activation particles were used to establish an in vitro model of overexpression of the bmal1 gene. Results: Melatonin promoted apoptosis of RAW264.7 cells and increased the expression of BMAL1 to inhibit the activation of ROS and phosphorylation of mitogen-activated protein kinase (MAPK)-p38. Silencing the bmal1 gene weakened the above effects of melatonin. After that, we used dehydrocorydaline (DHC) to enhance the activation of MAPK-p38, and the effects of melatonin on reducing ROS levels and promoting apoptosis of RAW264.7 cells were also blocked. Then, we constructed a mouse model of postmenopausal osteoporosis and administered melatonin. The results showed that melatonin improves bone loss in ovariectomized mice. Finally, we established a model of overexpression of the bmal1 gene, and these results suggest that the bmal1 gene can regulate ROS activity and change the level of the MAPK-p38 signalling pathway. Conclusion: Our study confirmed that melatonin promotes the apoptosis of RAW264.7 cells through BMAL1/ROS/MAPK-p38, and revealed the therapeutic effect and mechanism of melatonin in postmenopausal osteoporosis. This finding enriches BMAL1 as a potential target for the treatment of osteoporosis and the pathogenesis of postmenopausal osteoporosis. Cite this article: Bone Joint Res 2023;12(11):677–690.

Abstract Objective Differentiation of immortalized Mesenchymal Stromal Cells (iMSCs) into PDGFRα-positive cells under controlled growth conditions has several vital implications in functional studies concerned with the pathogenesis of Diabetic Gastroparesis (DGP). A study published previously by our research group demonstrated the importance of these cells as a novel, in-vitro model for investigating the functional role of neuronal nitric oxide synthase. The currently available methods require fresh differentiation of PDGFRα-positive cells for each round of experimentation. This leads to longer delays, higher usage of reagents, and inconsistency in reproducibility of experiments frequently. We thus aimed to establish through validation that cryopreserving and maintaining the iMSC-derived PDGFRα-positive cells for functional investigations help us to overcome these challenges. Results We demonstrated for the first time that the differentiated PDGFRα-positive cells from iMSCs can be cryopreserved and thawed to be used as per the experimental requirements with prolonged preservation of their characteristics. We assessed the viability of differentiated PDGFRα-positive cells pre- and post-freezing with the subsequent validation of their functional features using flow cytometry, qRT-PCR, and western blotting. We have been successful in demonstrating for the first time that the cryopreservation of previously differentiated PDGFRα-positive cells can be used as a feasible and cost-effective model for experimental reproducibility in functional studies of Diabetes Gastroparesis. Graphical Abstract

Abstract Background Current solutions for the analysis of Western Blot images lack either transparency and reproducibility or can be tedious to use if one has to ensure the reproducibility of the analysis. Results Here, we present an open-source gel image analysis program, IOCBIO Gel. It is designed to simplify image analysis and link the analysis results with the metadata describing the measurements. The software runs on all major desktop operating systems. It allows one to use it in either a single-researcher environment with local storage of the data or in a multiple-researcher environment using a central database to facilitate data sharing within the research team and beyond. By recording the original image and all operations performed on it, such as image cropping, subtraction of background, sample lane selection, and integration boundaries, the software ensures the reproducibility of the analysis and simplifies making corrections at any stage of the research. The analysis results are available either through direct access to the database used to store it or through the export of the relevant data. Conclusions The software is not only limited to Western Blot image analysis and can be used to analyze images obtained as a part of many other widely used biochemical techniques such as isoelectric focusing. By recording the original data and all the analysis steps, the program improves reproducibility in the analysis and contributes to the implementation of FAIR principles in the related fields.