Divide-and-Conquer Matrisome Protein (DC-MaP) Strategy: An MS-Friendly Approach to Proteomic Matrisome Characterization

Currently, the extracellular matrix (ECM) is considered a pivotal complex meshwork of macromolecules playing a plethora of biomolecular functions in health and disease beyond its commonly known mechanical role. Only by unraveling its composition can we leverage related tissue engineering and pharmacological efforts. Nevertheless, its unbiased proteomic identification still encounters some limitations mainly due to partial ECM enrichment by precipitation, sequential fractionation using unfriendly-mass spectrometry (MS) detergents, and resuspension with harsh reagents that need to be entirely removed prior to analysis. These methods can be technically challenging and labor-intensive, which affects the reproducibility of ECM identification and induces protein loss. Here, we present a simple new method applicable to tissue fragments of 10 mg and more. The technique has been validated on human ovarian tissue and involves a standardized procedure for sample processing with an MS-compatible detergent and combined centrifugation. This two-step protocol eliminates the need for laborious sample clarification and divides our samples into 2 fractions, soluble and insoluble, successively enriched with matrisome-associated (ECM-interacting) and core matrisome (structural ECM) proteins.     

Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection

In this paper, the development of the Paphiopedilum Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of P. Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The P. Maudiae embryo sac exhibited an Allium type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50–54 DAP. In planta transformation was achieved by injection of the ovaries by Agrobacterium, resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the hpt gene in the leaves of eight hygromycin-resistant seedlings following Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while hpt expression was not detected in the control. The best time to inject P. Maudiae ovaries in planta with Agrobacterium is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any Paphiopedilum species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics.     

Molecular Aspects and Prognostic Significance of Microcalcifications in Human Pathology: A Narrative Review

The presence of calcium deposits in human lesions is largely used as imaging biomarkers of human diseases such as breast cancer. Indeed, the presence of micro- or macrocalcifications is frequently associated with the development of both benign and malignant lesions. Nevertheless, the molecular mechanisms involved in the formation of these calcium deposits, as well as the prognostic significance of their presence in human tissues, have not been completely elucidated. Therefore, a better characterization of the biological process related to the formation of calcifications in different tissues and organs, as well as the understanding of the prognostic significance of the presence of these calcium deposits into human tissues could significantly improve the management of patients characterized by microcalcifications associated lesions. Starting from these considerations, this narrative review highlights the most recent histopathological and molecular data concerning the formation of calcifications in breast, thyroid, lung, and ovarian diseases. Evidence reported here could deeply change the current point of view concerning the role of ectopic calcifications in the progression of human diseases and also in the patients’ management. In fact, the presence of calcifications can suggest an unfavorable prognosis due to dysregulation of normal tissues homeostasis.     

Recent Update on the Molecular Mechanisms of Gonadal Steroids Action in Adipose Tissue

The gonadal steroids, including androgens, estrogens and progestogens, are involved in the control of body fat distribution in humans. Nevertheless, not only the size and localization of the fat depots depend on the sex steroids levels, but they can also highly affect the functioning of adipose tissue. Namely, the gonadocorticoids can directly influence insulin signaling, lipid metabolism, fatty acid uptake and adipokine production. They may also alter energy balance and glucose homeostasis in adipocytes in an indirect way, e.g., by changing the expression level of aquaglyceroporins. This work presents the recent advances in understanding the molecular mechanism of how the gonadal steroids influence the functioning of adipose tissue leading to a set of detrimental metabolic consequences. Special attention is given here to highlighting the sexual dimorphism of adipocyte functioning in terms of health and disease. Particularly, we discuss the molecular background of metabolic disturbances occurring in consequence of hormonal imbalance which is characteristic of some common endocrinopathies such as the polycystic ovary syndrome. From this perspective, we highlight the potential drug targets and the active substances which can be used in personalized sex-specific management of metabolic diseases, in accord with the patient’s hormonal status.     

Role of the central and peripheral nervous system in the ovarian function

This review attempts to give a comprehensive overview of ovarian innervation, considering the whole nervous system and its different levels that may modify the ovarian function. The connection between the ovary and the central nervous system through the autonomic pathways, including the peripheral ganglia, is highlighted. The evidence obtained over the last years highlights the role of the superior ovarian nerve (SON) in the ovarian phenomena. Besides, the effect on the ovary of conventional neurotransmitters and others such as indolamines and peptides, which have been found in this organ, are discussed. Various reproductive diseases have been studied almost exclusively from the endocrine point of view. It is evident that a better knowledge about the role of the neural factors involved in the ovarian physiology may facilitate the understanding of some of these. A review of the concepts and an update of some experimental designs is made that permits clarifying several aspects of the relationship between the neural system and the ovary. At present, there is no doubt that the innervation of the ovary is involved in several physiological aspects of this gland function. However, the relationship of some levels of the nervous system and the ovary offer a wide avenue for future research.     

Cell cycle analysis of Chinese hamster ovary cells stimulated by phosphatidic acid in serum-free culture

When recombinant Chinese hamster ovary cells were treated with pertussis toxin or genistein, not only lysophosphatidic acid (LPA) but also phosphatidic acid (PA) failed to stimulate progression through the cell cycle in serum-free culture, suggesting that PA and LPA induce cell growth through the same signal transduction pathway. Cell cycle analysis also indicates that cell growth promoted by PA results in enhanced protein production.     

PPARγ基因外显子6多态性与PCOS的相关性研究

目的探讨过氧化物酶体增殖物激活受体7（PPAR7）基因外显子6多态性与多囊卵巢综合征（PCOS）的关系。方法抽取中国汉族111例PCOS患者（PCOS组）和92例非DCOS女性患者（对照组）静脉血;特异引物聚合酶链反应（PCR）扩增后限制性酶切（PCR—RFLP）,电泳分离,检测PPARγ基因外显子6的多态性,并分析其与P—COS患者各项临床特征的关系。结果PCOS组中CT／TT基因型频率、T等位基因频率明显高于对照组（P〈0．01）;PCOS患者中PPARγ基因外显子6突变者BMI、腰围、臀围、空腹血糖明显高于未突变者（P〈0．05）,肥胖型PCOS患者中突变（CT／TT）明显高于非肥胖型PCOS患者（P〈0．05）。结论PPAR7基因外显子6的多态性与PCOS肥胖的发生、空腹血糖升高有关。     

Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells

Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, primarily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an increased glutamate concentration on cell metabolism and rAT production were investigated. When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maximum lactate concentration decreased with decreasing pH. The highest production level of rAT was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glucose consumption rate (q(Glc)) with increasing culture time was observed in all the above-mentioned experiments, while the glucose concentration was maintained above 0.7 g l(-1). In addition, a decrease in the specific rAT production rate (q(rAT)) was observed after the depletion of lactate in the late cultivation stage. Taken together, these results suggest that the reduced availability of cellular energy caused by the decrease in q(Glc) and depletion of lactate led to the decrease in q(rAT). This decrease in q(rAT) was partially prevented by increasing the residual glutamate concentration from 1 mM to 7 mM, thus resulting in an additional 30% increase in the amount of rAT activity. The optimized fed-batch culture process yielded 1.0 g l(-1) rAT at 287 h of cultivation.     

美洲鲥鱼卵巢发育规律和性类固醇激素变化研究

采用组织学、形态学及电化学等技术方法,研究了人工养殖条件下美洲鲥鱼雌性亲鱼卵巢发育和性类固醇激素的周期变化规律。结果表明,美洲鲥鱼属于非同步分批产卵类型;根据卵巢外部形状大小及色泽等的不同,可将美洲鲥鱼卵巢发育过程分为6个时期;根据卵母细胞的形态结构、卵黄物质的积累和滤泡细胞的变化,将卵子发生划分为6个时相;卵巢周期变化过程中,肝脏指数(HSI)和肥满度(CF)变化不显著,性腺指数(GSI)与血浆中雌二醇(E2)水平表现出与性腺发育协同一致的变化规律。