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Biomedicines May 2024Today, women's infertility is considered a social disease in females, occurring not only as an effect of POF (premature ovarian failure) but also as CTRI (cancer... (Review)
Review
Today, women's infertility is considered a social disease in females, occurring not only as an effect of POF (premature ovarian failure) but also as CTRI (cancer treatment-related infertility) in oncologic patients. Several procedures for FP (fertility preservation) are currently adopted to prevent this condition, mostly based on utilization of retrieved eggs from the patients with subsequent IVF (in vitro fertilization) or cryopreservation. However, great interest has recently been devoted to OSCs (ovarian stem cells), whose isolation from female ovaries, followed by their in vitro culture, led to their maturation to OLCs (oocyte-like cells), namely, neo-oocytes comparable to viable eggs suitable for IVF. Translation of these data to FP clinical application creates new hope in the treatment of infertility. Thus, in line with the significant progress in using stem cells in the regenerative medicine field, neo-oogenesis via OSCs, which is currently unapplicable in fertility preservation procedures, will provide novel possibilities for young and adult females in motherhood programs in the future.
PubMed: 38927346
DOI: 10.3390/biomedicines12061139 -
BMC Biotechnology Jun 2024Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with...
BACKGROUND
Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS
In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS
The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS
This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Topics: Animals; CHO Cells; Integrases; Cricetulus; Nuclear Localization Signals; Cell Nucleus; Serine; Green Fluorescent Proteins; Cricetinae; Xenopus laevis
PubMed: 38926833
DOI: 10.1186/s12896-024-00871-4 -
BMC Veterinary Research Jun 2024The present study was performed to characterize and compare the perfusion of vaginal and uterine arteries after challenging the reproductive tract of dairy cows via...
AIM
The present study was performed to characterize and compare the perfusion of vaginal and uterine arteries after challenging the reproductive tract of dairy cows via natural mating, artificial insemination (AI), or intravaginal deposition (vaginal fundus) of different biological fluids or a placebo.
MATERIALS AND METHODS
In a double-blind study, six German Holstein cows were administered PGF during dioestrus and 48 h later treated with GnRH. Intravaginal or intrauterine treatments were carried out 12 h after GnRH was administered. Animals served as their controls, using a cross-over design with an interval of 14 days between experiments. The experimental animals were allocated to receive the following treatments: natural mating (N), intrauterine artificial insemination (A), intravaginal deposition (vaginal fundus) of 6 mL raw semen (R) or 6 mL seminal plasma (S), and compared to their controls [control 1: 6 mL placebo (P: physiological saline); control 2: no treatment (C)). Corresponding time intervals were chosen for the untreated control oestrus. Blood flow volume (BFV) in the uterine (u) and vaginal (v) arteries ipsilateral to the ovary bearing the preovulatory follicle was determined using transrectal Doppler sonography.
RESULTS
All animals exhibited oestrus and ovulated between 30 and 36 h after GnRH. Transient increases (P < 0.05) in vaginal blood flow occurred between 3 and 12 h following mating as well as 3 to 9 h after deposition of raw semen and seminal plasma, respectively. The most distinct increases (199%) in vBFV occurred 6 h after mating compared to values immediately before mating (= time 0 h). Neither AI nor deposition of a placebo into the vagina affected vBFV (P > 0.05). Only mating and deposition of either raw semen, seminal plasma or AI increased uBFV (P < 0.003). The greatest rise in uBFV occurred after natural mating. Maximum uBFV values were detected 9 h after mating when values were 79% greater (P < 0.05) than at 0 h.
CONCLUSIONS
The natural mating, deposition of raw semen or seminal plasma and conventional AI affect vaginal and/or uterine blood flow to different degrees. The factors responsible for these alterations in blood flow and their effects on fertility remain to be clarified in future studies.
Topics: Animals; Insemination, Artificial; Female; Semen; Cattle; Vagina; Uterus; Male; Administration, Intravaginal; Double-Blind Method; Gonadotropin-Releasing Hormone; Cross-Over Studies; Regional Blood Flow
PubMed: 38926710
DOI: 10.1186/s12917-024-03919-x -
BMC Biology Jun 2024The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis,...
BACKGROUND
The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii, and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems.
RESULTS
We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex-determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplicates (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been likely lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome 18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variations (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex-determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in the testis than in the ovary.
CONCLUSIONS
Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.
Topics: Animals; Sex Determination Processes; Evolution, Molecular; Male; Female; Perches; Phylogeny; Receptors, Peptide; Genome; Receptors, Transforming Growth Factor beta
PubMed: 38926709
DOI: 10.1186/s12915-024-01935-9 -
Scientific Reports Jun 2024Previously, we demonstrated the expression of visfatin in porcine reproductive tissues and its effect on pituitary endocrinology. The objective of this study was to...
Previously, we demonstrated the expression of visfatin in porcine reproductive tissues and its effect on pituitary endocrinology. The objective of this study was to examine the visfatin effect on the secretion of steroid (P, E) and prostaglandin (PGE, PGF), the mRNA and protein abundance of steroidogenic markers (STAR, CYP11A1, HSD3B, CYP19A1), prostaglandin receptors (PTGER2, PTGFR), insulin receptor (INSR), and activity of kinases (MAPK/ERK1/2, AKT, AMPK) in the porcine corpus luteum. We noted that the visfatin effect strongly depends on the phase of the estrous cycle: on days 2-3 and 14-16 it reduced P, while on days 10-12 it stimulated P. Visfatin increased secretion of E on days 2-3, PGE on days 2-3 and 10-12, reduced PGF release on days 14-16, as well as stimulated the expression of steroidogenic markers on days 10-12 of the estrous cycle. Moreover, visfatin elevated PTGER mRNA expression and decreased its protein level, while we noted the opposite changes for PTGFR. Additionally, visfatin activated ERK1/2, AKT, and AMPK, while reduced INSR phosphorylation. Interestingly, after inhibition of INSR and signalling pathways visfatin action was abolished. These findings suggest a regulatory role of visfatin in the porcine corpus luteum.
Topics: Animals; Corpus Luteum; Female; Swine; Nicotinamide Phosphoribosyltransferase; Estrous Cycle; Receptor, Insulin; Progesterone; Receptors, Prostaglandin; Dinoprost
PubMed: 38926439
DOI: 10.1038/s41598-024-65102-4 -
NPJ Vaccines Jun 2024Following administration of the SARS-CoV-2 vaccine, many women worldwide reported short-term menstrual irregularities. Although menstrual bleeding, "the fifth vital...
Following administration of the SARS-CoV-2 vaccine, many women worldwide reported short-term menstrual irregularities. Although menstrual bleeding, "the fifth vital sign", is experienced by more than 300 million people on any given day worldwide, these changes were only partially studied. Irregular periods are important well beyond fertility and the discomfort they impose; they are associated with the risk of cardiovascular morbidity, chronic diseases, and premature mortality. Pre-clinical examination of the vaccine polymeric envelope indicates its accumulation in the ovaries. The somatic endocrine cells of the ovarian follicle - the granulosa cells (GCs)-participate in the strict hypothalamic-pituitary-ovarian (HPO) feedback loop that governs the menstrual cycle via endocrine and paracrine regulators, as AMH and Inhibins. We aimed to unravel the direct effect of the COVID-19 vaccine on GCs and link their post-vaccine activity to changes in menstrual patterns. Human primary GCs exposed in-vitro to the Pfizer COVID-19 vaccine BNT162b2, demonstrated no change in their viability but altered mRNA transcripts, specifically of the regulatory key factors: InhibinB was upregulated, whereas AMH was downregulated. We further examined pre- and post-vaccination blood samples from individual women and found a 2-3 folds change in the post-vaccination FSH/InhibinB protein level ratio, compared to their pre-vaccination values. This altered expression of InhibinB could significantly impact the HPO axis in vaccinated women and may ultimately influence the endometrium cyclicity, manifested clinically by the commonly reported changes in menstrual bleeding patterns.
PubMed: 38926432
DOI: 10.1038/s41541-024-00911-2 -
Cancer Medicine Jun 2024Accurate characterization of newly diagnosed a solid adnexal lesion is a key step in defining the most appropriate therapeutic approach. Despite guidance from the...
BACKGROUND
Accurate characterization of newly diagnosed a solid adnexal lesion is a key step in defining the most appropriate therapeutic approach. Despite guidance from the International Ovarian Tumor Analyzes Panel, the evaluation of these lesions can be challenging. Recent studies have demonstrated how machine learning techniques can be applied to clinical data to solve this diagnostic problem. However, ML models can often consider as black-boxes due to the difficulty of understanding the decision-making process used by the algorithm to obtain a specific result.
AIMS
For this purpose, we propose an Explainable Artificial Intelligence model trained on clinical characteristics and qualitative ultrasound indicators to predict solid adnexal masses diagnosis.
MATERIALS & METHODS
Since the diagnostic task was a three-class problem (benign tumor, invasive cancer, or ovarian metastasis), we proposed a waterfall classification model: a first model was trained and validated to discriminate benign versus malignant, a second model was trained to distinguish nonmetastatic versus metastatic malignant lesion which occurs when a patient is predicted to be malignant by the first model. Firstly, a stepwise feature selection procedure was implemented. The classification performances were validated on Leave One Out scheme.
RESULTS
The accuracy of the three-class model reaches an overall accuracy of 86.36%, and the precision per-class of the benign, nonmetastatic malignant, and metastatic malignant classes were 86.96%, 87.27%, and 77.78%, respectively.
DISCUSSION
SHapley Additive exPlanations were performed to visually show how the machine learning model made a specific decision. For each patient, the SHAP values expressed how each characteristic contributed to the classification result. Such information represents an added value for the clinical usability of a diagnostic system.
CONCLUSIONS
This is the first work that attempts to design an explainable machine-learning tool for the histological diagnosis of solid masses of the ovary.
Topics: Humans; Female; Machine Learning; Ultrasonography; Ovarian Neoplasms; Middle Aged; Adult; Adnexal Diseases; Aged; Algorithms; Diagnosis, Differential
PubMed: 38923847
DOI: 10.1002/cam4.7425 -
Veterinary Sciences Jun 2024The quality of animal feed is increasingly affected by weather conditions, high humidity, and damage to grains, which have led to various mycotoxin-producing moulds. The...
The quality of animal feed is increasingly affected by weather conditions, high humidity, and damage to grains, which have led to various mycotoxin-producing moulds. The aim of this study was to determine the effects of the combination of deepoxy-deoxynivalenol and beta-zearalenol on the development of preimplantation bovine embryos, the extent to which the presence of both mycotoxin metabolites affects the development of in vitro cultured bovine embryos, or whether the effect of both toxins enhances embryotoxicity. Ovaries were transported from the abattoir to the laboratory and, after maturation and fertilisation, zygotes were placed in an embryo culture medium (IVC) with different mycotoxin metabolite concentrations diluted in acetonitrile. It was found that the blastocyst rate of cleaved embryos was affected by 1 μL acetonitrile in 400 μL medium (0.25%) compared to the group without acetonitrile. For this reason, it was decided to use acetonitrile as a control group, and the desired mycotoxin metabolite concentrations were diluted in the lowest possible amount of acetonitrile (0.5 μL) that could be accurately added to the study groups. There was no statistical difference when the higher mycotoxin metabolite concentrations were added.
PubMed: 38922014
DOI: 10.3390/vetsci11060267 -
Veterinary Sciences May 2024Cavitary corpora lutea are commonly observed during the estrous cycle in bovines. Since the quality of the corpus luteum (CL) is extremely important during embryo...
Cavitary corpora lutea are commonly observed during the estrous cycle in bovines. Since the quality of the corpus luteum (CL) is extremely important during embryo transfer when embryos are implanted into the recipient, the ultrasonographic examination of the CL is becoming more and more important in terms of the outcome of the procedure. In the present study, a total of 2477 ultrasonographic transrectal diagnoses were performed, and data were collected between the years of 2018 and 2020 in a large-scale Holstein Friesian dairy farm in Hungary. In 91.1% (n = 2257) and in 8.9% (n = 220) of the cases, compact CLs and cavitary CLs, respectively, were diagnosed at pregnancy diagnosis. The presence of a cavitary CL on the ovary at pregnancy diagnosis increased the odds of remaining open after pregnancy by 21 times compared to the presence of a compact CL (OR = 21.0, < 0.001) in the cows. The presence of cavitary CL was not influenced either by month or season. Ovarian cysts were detected in 196 cases (8.0%) in the examined animals. The presence of a cavitary CL decreased by 9 times when an ovarian cyst was also diagnosed (OR = 9.0, 1.6% vs. 9.5%, < 0.001). The presence of an ovarian cyst decreased the odds of established pregnancy by 81 times (OR = 81.1, < 0.001). Based on our results, the presence of a cavitary CL between days 31 and 42 after artificial insemination is associated with a smaller chance of conception in Holstein Friesian cows. The presence of an ovarian cyst decreases the occurrence of cavitary CL and the chance of conception.
PubMed: 38921993
DOI: 10.3390/vetsci11060246 -
Insects Jun 2024Death-associated protein-1 (DAP-1) plays a crucial role in cell growth, migration, autophagy, and apoptosis in mammals. However, its function in insects remains unclear....
Death-associated protein-1 (DAP-1) plays a crucial role in cell growth, migration, autophagy, and apoptosis in mammals. However, its function in insects remains unclear. In the present study, we cloned and identified DAP-1 (NlDAP-1). NlDAP-1 was expressed during all developmental stages and in all tissues of , being particularly higher in the ovaries of female adults. RNAi with double-stranded NlDAP-1 RNA significantly inhibited the expression of NlDAP-1, leading to premature death (dying seven days earlier), delayed ovarian development, and fewer offspring (76.7% reduction in eggs with 77.4% reduction in egg hatching rate). Additionally, an immunofluorescence experiment showed that NlDAP-1 was highly expressed when yeast-like symbionts (YLSs) entered oocytes, and inhibiting the expression of NlDAP-1 disturbed the process; the RNAi of NlDAP-1 caused a 34.9% reduction in the YLSs that entered oocytes. These results indicate that NlDAP-1 plays a crucial role in the reproductive development of and the transovarial transmission of its YLSs.
PubMed: 38921140
DOI: 10.3390/insects15060425