NRF2 attenuation aggravates detrimental consequences of metabolic stress on cultured porcine parthenote embryos.

The nuclear factor erythroid 2-related factor 2 (NRF2) is a crucial transcription factor that plays a central role in regulating oxidative stress pathways by binding antioxidant response elements, but its involvement in early embryo development remains largely unexplored. In this study, we demonstrated that NRF2 mRNA is expressed in porcine embryos from day 2 to day 7 of development, showing a decrease in abundance from day 2 to day 3, followed by an increase on day 5 and day 7. Comparable levels of NRF2 mRNA were observed between early-cleaving and more developmental competent embryos and late-cleaving and less developmental competent embryos on day 4 and day 5 of culture. Attenuation of NRF2 mRNA significantly decreased development of parthenote embryos to the blastocyst stage. When NRF2-attenuated embryos were cultured in presence of 3.5 mM or 7 mM glucose, development to the blastocyst stage was dramatically decreased in comparison to the control group (15.9% vs. 27.8% for 3.5 mM glucose, and 5.4% vs. 25.3% for 7 mM glucose). Supplementation of melatonin moderately improved the development of NRF2-attenuated embryos cultured in presence of 0.6 mM glucose. These findings highlight the importance of NRF2 in early embryo development, particularly in embryos cultured under metabolically stressful conditions.

Severe body condition loss lowers hepatic output of IGF1 with adverse effects on the dominant follicle in dairy cows.

The severe loss of body condition score (BCS) during the early lactation period has been associated with infertility in cows. However, the mechanisms are not fully understood. The aim of this study was to examine the effect of BCS loss on liver health, and ovarian functions in cows during early lactation. Retrospectively multiparous cows from two farms were categorized based on units of BCS (1-5 scale) loss as Moderate (MOD, <0.75 units; n = 11) or Severe (SEV, ≥0.75 units; n = 9) loss groups. From Weeks -3 to 7, relative to calving, MOD and SEV cows lost on average 0.4 and 1.0-unit BCS, respectively. All data except hepatic transcriptomes were analyzed with PROC MIXED procedure of SAS. The plasma concentration of non-esterified fatty acids at Week 0 and 1, ß-hydroxy butyrate at Week 1, and γ-glutamyl transferase at Weeks 1 and 7 relative to calving were higher in SEV cows. Hepatic transcriptome analysis showed that 1 186 genes were differentially expressed in SEV (n = 3) compared to MOD (n = 3) cows at Week 7 after calving. Pathway analysis revealed that significant DEGs in SEV cows enriched in lipid metabolisms including, lipid metabolic process, ether lipid metabolism, fatty acid beta-oxidation, fatty acid biosynthetic process, fatty acid metabolic process, fat digestion and absorption, linoleic acid metabolism, alpha-linolenic acid metabolism. The impaired liver function in SEV cows was associated with 1.5-fold reduction of hepatic IGF1 gene expression and lower serum IGF1 concentrations. At the ovarian level, SEV cows had lower IGF1 concentration in the follicular fluid of the dominant follicle of the synchronized follicular wave compared to that of MOD cows at 7 weeks after calving. Further, the follicular fluid concentration of estradiol-17ß was lower in SEV cows along with lower transcript abundance of genes from granulosa cells associated with dominant follicle competence, including CYP19A1, NR5A2, IGF1, and LHCGR. These data show that SEV loss of BCS during early lactation leading up to the planned start of breeding is associated with liver dysfunction, including lower IGF1 secretion, and impaired function of the dominant follicle in the ovary.

Primary Peritoneal Carcinosarcoma in a Breast Cancer Patient Harboring a Germline BRCA2 Pathogenic Variant: Case Report.

Malignant mixed müllerian tumor (MMMT) is a rare neoplasm, consisting of carcinomatous (epithelial) and sarcomatous (mesenchymal) components that most commonly arise in the endometrium and more infrequently in the ovaries, fallopian tube, cervix, and vagina. Primary peritoneal carcinosarcoma (PPCS) is an extremely rare extragenital presentation of MMMT. Although the occurrence of breast cancer and epithelial ovarian carcinoma in association with BRCA pathogenic variants is firmly established, the etiologic role of these genes in the development of other tumor types is less well known. Here, we present a rare case of PPCS in a 42-year-old Brazilian woman with a BRCA2 pathogenic variant, c.2808_2811del (NM_000059.3). The patient developed metastatic breast cancer at the age of 37 and underwent a risk-reducing bilateral salpingo-oophorectomy 2 years later. She was then diagnosed with PPCS 3 years after the risk-reducing surgery. She underwent treatment with surgery, chemotherapy, and targeted therapy but passed away almost 5 years after the second primary tumor diagnosis. To our knowledge, this is the first case of peritoneal carcinosarcoma described in a BRCA2 pathogenic variant carrier, and its report leads to a better understanding of the disease's molecular features and possible therapeutic approaches.

DCAF13 and RNF114 participate in the regulation of early porcine embryo development.

In brief: Ubiquitination plays a pivotal role in a multitude of cellular functions; however, the precise contributions of various ubiquitin ligases in governing early developmental processes remain largely unexplored. This study revealed that the E3 ubiquitin ligases DCAF13 and RNF114 are both necessary for the normal regulation of early porcine embryo development. Abstract: Ubiquitylation is required for normal regulation of many biological functions by modulating several protein facets such as structure, stability, interaction, localization, and degradation. In this study, we explored the roles of two E3 ubiquitin ligases (E3s), the DDB1- and CUL4-associated factor 13 (DCAF13) and the Ring finger protein 114 (RNF114), in the regulation of porcine embryo development. Attenuation of DCAF13 mRNA decreased embryo development at the blastocyst stage, while the development of RNF114-attenuated embryos was not significantly different than that of control embryos. The average number of cells per blastocyst was decreased in DCAF13-attenuated embryos and increased in RNF114-attenuated embryos compared to controls. The relative mRNA abundance of the histone methyltransferase SUV39H1, which regulates histone H3 lysine 9 trimethylation (H3K9me3), was increased in both DCAF13- and RNF114-attenuated embryos, but nuclear immunofluorescence signal for H3K9me3 on day 3 embryos was not significantly altered between attenuated and control embryos. Nuclear immunofluorescence signal for H3K4m3 was decreased in DCAF13-attenuated embryos, but it was increased in RNF114-attenuated embryos compared to controls. Attenuation of DCAF13 and RNF114 mRNAs increased transcript levels for the DNA recombinase RAD51 and decreased expression of phosphorylated histone H2A.X (γH2AX), which suggests an impact on DNA damage repair. In addition, lower mRNA expression of the lysine demethylases 5B (KDM5B) and 5C (KDM5C), both involved in embryo genome activation and DNA repair, was detected in DCAF13-attenuated embryos. These findings indicated that both DCAF13 and RNF114 have important roles in the regulation of the early development of porcine embryos.

The Clinical and Molecular Profile of Lung Cancer Patients Harboring the TP53 R337H Germline Variant in a Brazilian Cancer Center: The Possible Mechanism of Carcinogenesis.

In southern and southeastern Brazil, the TP53 founder variant c.1010G>A (R337H) has been previously documented with a prevalence of 0.3% within the general population and linked to a heightened incidence of lung adenocarcinomas (LUADs). In the present investigation, we cover clinical and molecular characterizations of lung cancer patients from the Brazilian Li-Fraumeni Syndrome Study (BLISS) database. Among the 175 diagnosed malignant neoplasms, 28 (16%) were classified as LUADs, predominantly occurring in females (68%), aged above 50 years, and never-smokers (78.6%). Significantly, LUADs manifested as the initial clinical presentation of Li-Fraumeni Syndrome in 78.6% of cases. Molecular profiling was available for 20 patients, with 14 (70%) revealing EGFR family alterations. In total, 23 alterations in cancer driver genes were identified, comprising 7 actionable mutations and 4 linked to resistance against systemic treatments. In conclusion, the carriers of TP53 R337H demonstrate a predisposition to LUAD development. Furthermore, our results indicate that environmental pollution potentially impacts the carcinogenesis of lung tumors in the carriers of TP53 R337H.

Brazilian Expert Consensus for NTRK Gene Fusion Testing in Solid Tumors.

Oncogenic neurotrophic tropomyosin receptor kinase gene fusions occur in less than 1% of common cancers. These mutations have emerged as new biomarkers in cancer genomic profiling with the approval of selective drugs against tropomyosin receptor kinase fusion proteins. Nevertheless, the optimal pathways and diagnostic platforms for this biomarker's screening and genomic profiling have not been defined and remain a subject of debate. A panel of national experts in molecular cancer diagnosis and treatment was convened by videoconference and suggested topics to be addressed in the literature review. The authors proposed a testing algorithm for oncogenic neurotrophic tropomyosin receptor kinase gene fusion screening and diagnosis for the Brazilian health system. This review aims to discuss the latest literature evidence and international consensus on neurotrophic tropomyosin receptor kinase gene fusion diagnosis to devise clinical guidelines for testing this biomarker. We propose an algorithm in which testing for this biomarker should be requested to diagnose advanced metastatic tumors without known driver mutations. In this strategy, Immunohistochemistry should be used as a screening test followed by confirmatory next-generation sequencing in immunohistochemistry-positive cases.

Multiplatform Analysis of Intratumoral PTEN Heterogeneity in Melanoma.

Loss of protein expression of the tumor suppressor PTEN is associated with increased cancer aggressiveness, decreased tumor immune infiltration, and resistance to immune and targeted therapies in melanoma. We assessed a unique cohort of eight melanoma samples with focal loss of PTEN protein expression to understand the features and mechanisms of PTEN loss in this disease. We compared the PTEN-negative (PTEN[-]) areas to their adjacent PTEN-positive (PTEN[+]) areas using DNA sequencing, DNA methylation, RNA expression, digital spatial profiling, and immunohistochemical platforms. Variations or homozygous deletions of PTEN were identified in PTEN(-) areas that were not detected in the adjacent PTEN(+) areas in three cases (37.5%), but no clear genomic or DNA methylation basis for loss was identified in the remaining PTEN(-) samples. RNA expression data from two independent platforms identified a consistent increase in chromosome segregation gene expression in PTEN(-) versus adjacent PTEN(+) areas. Proteomic analysis showed a relative paucity of tumor-infiltrating lymphocytes in PTEN(-) versus adjacent PTEN(+) areas. The findings add to our understanding of potential molecular intratumoral heterogeneity in melanoma and the features associated with the loss of PTEN protein in this disease.

Optimization of gonadotropin stimulation protocols for in vitro embryo production in prepubertal Mediterranean water buffalo.

Embryos can be produced from prepubertal donor animals using laparoscopic ovum pickup and in vitro embryo production technologies (LOPU-IVEP). Together, these tools can shorten the interval between generations, rapidly accelerating the rate of genetic gain. Here, we assessed the impact of different gonadotropin stimulation protocols in Mediterranean water buffalo heifer calves aged between 2 and 6 months old. Following gonadotropin stimulation, LOPU was performed at two-week intervals, with animals receiving different protocols on subsequent LOPUs. After collection, the cumulus-oocyte complexes (COCs) were matured and fertilized in vitro, and embryos were cultured to the blastocyst stage followed by transfer into synchronized adult recipients. The number and size of follicles aspirated during LOPU, the number and quality of COCs recovered, as well as cleavage, embryo development and pregnancy rates were assessed. First, we evaluated the impact of using FSH with and without eCG (administered 24-h prior to LOPU) and found that a combination of FSH and eCG was able to significantly improve embryo development rates (20.6 ± 2.0% vs. 9.0 ± 3.6%; P < 0.05). Second, we compared this protocol to a slow-release formulation of FSH reconstituted in hyaluronan. In addition to requiring less work to prepare the animals for LOPU, this slow-release formulation yielded numerically higher, but not statistically different, average number of recovered COCs (14.4 ± 2.1 vs. 10.3 ± 2.0; P > 0.05) and embryo development rates (22.9 ± 4.7% vs. 14.1 ± 5.2%; P > 0.05) compared to FSH given every 12 h. Next, we compared the length of gonadotropin treatment over 3-, 4- and 5-days prior to LOPU and found that as the length of gonadotropin treatment increased, although the number of COCs recovered steadily decreased (14.1 ± 2.4 vs. 8.7 ± 1.0 vs. 6.9 ± 0.7; P < 0.05), the embryo development rates steadily increased (14.4 ± 3.9 vs. 27.3 ± 4.4 vs. 35.9 ± 7.0; P < 0.05), presumably due to an increase in the proportion of large follicles at the time of LOPU. Numerically, the 4-day treatment yielded more transferrable embryos per donor per LOPU (2.70 ± 0.5) than 3-day (1.94 ± 0.6) and 5-day (2.25 ± 0.5) treatments. Finally, following embryo transfer, 26 of 90 recipient females became pregnant (28.9%). Pregnancies were established from all treatments, which suggests that post-implantation development was not affected among the gonadotropin treatments assessed.

Tumor glycolytic profiling through 18F-FDG PET/CT predicts immune checkpoint inhibitor efficacy in advanced NSCLC.

Background: A significant proportion of patients with non-small-cell lung cancer (NSCLC) do not respond to immune checkpoint inhibitors (ICIs). Since metabolic reprogramming with increased glycolysis is a hallmark of cancer and is involved in immune evasion, we used 18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG PET/CT) to evaluate the baseline glycolytic parameters of patients with advanced NSCLC submitted to ICIs, and assessed their predictive value. Methods: 18F-FDG PET/CT results in the 3 months before ICIs treatment were included. Maximum standardized uptake values, whole metabolic tumor volume (wMTV), and whole-body total lesion glycolysis (wTLG) were evaluated. Cutoff values for high or low glycolytic categories were determined using receiver-operating characteristic curves. Progression-free survival (PFS) and overall survival (OS) were evaluated. Patients with a complete response and a matching group with resistance to ICIs underwent immunohistochemistry analysis. An unsupervised k-means clustering model integrating programmed cell death ligand 1 (PD-L1) expression, glycolytic parameters, and ICIs therapy was performed. Results: In all, 98 patients were included. Lower baseline 18F-FDG PET/CT parameters were associated with responses to ICIs. Patients with low wMTV or wTLG had improved PFS and OS. High wTLG, strong tumor expression of glucose transporter-1, and lack of responses were significantly associated. Patients with low glycolytic parameters benefited from ICIs, regardless of chemotherapy. Conversely, those with high parameters benefited from the addition of chemotherapy. Patients with higher wTLG and lower PD-L1 were associated with progression and worse survival to ICIs monotherapy. Conclusions: Glycolytic metabolic profiles established through baseline 18F-FDG PET/CT are useful biomarkers for evaluating ICI therapy in advanced NSCLC.

Factors Affecting the Efficiency of In Vitro Embryo Production in Prepubertal Mediterranean Water Buffalo.

Embryos from prepubertal water buffalo can be produced using laparoscopic ovum pickup (LOPU) and in vitro embryo production (IVEP). However, to date, it is unclear what factors and environmental conditions can affect LOPU-IVEP efficiency in prepubertal animals, especially buffalo. In this study, we explored the impact of season, age and individual variation among female donor animals, as well as the effect of the sire used for in vitro fertilization. Donor animals between 2 and 6 months of age were stimulated using gonadotropins prior to LOPU, which was performed at two-week intervals. Following in vitro maturation and fertilization, the resulting embryos were then cultured to the blastocyst stage until they were either vitrified or transferred into recipient animals. The number of follicles available for aspiration and embryo development rates was stable throughout the year. As animals became older, there was a slight trend for fewer COCs recovered from LOPU and better embryo development. There was a large individual variation in both ovarian response and the developmental competence of oocytes among donors. The bull used for fertilization also had a significant impact on embryo development. Upon embryo transfer, pregnancy rates were not affected by the number of embryos transferred per recipient. The best pregnancy rates were achieved when transferring blastocysts, compared to compact morula or hatched blastocysts. Finally, vitrification had no effect on pregnancy rate compared to fresh embryos.

Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

Optimizing swine in vitro embryo production with growth factor and antioxidant supplementation during oocyte maturation.

Porcine in vitro fertilization often results in low embryo development rates compared to other livestock species, which is often associated with either a low fertilization rate or high incidence of polyspermy. Since the quality of oocyte maturation is known to play a significant role in oocyte competence, we investigated the impact of supplementing in vitro maturation (IVM) medium containing porcine follicular fluid (pFF) with the growth factors FGF2, LIF and IGF1 (FLI), along with different combinations of cysteine, melatonin and ITS, on cumulus cell expansion, oocyte meiotic maturation, fertilization outcome, embryo development and blastocyst cell numbers. Maturation medium containing pFF yielded the greatest cumulus expansion. Compared to pFF and FLI individually, using pFF and FLI together resulted in the best embryo development rates over total oocyte placed in IVF (12.5% vs. 15.0% vs. 26.6%, respectively). Supplementation of IVM medium containing pFF and FLI with either cysteine, melatonin or insulin-transferrin-selenium, revealed that cysteine was essential to improve embryo development, while melatonin and ITS had a limited impact on improving blastocyst rates. Finally, we observed that pig oocytes matured in medium supplemented with pFF, FLI, cysteine and melatonin had a high proportion of monospermic zygotes (68.2%) and low proportion of polyspermic zygotes (15.9%) following IVF and yielded superior cleavage (78.2%) and blastocyst (32.0%) rates.

DNA Damage Induction Alters the Expression of Ubiquitin and SUMO Regulators in Preimplantation Stage Pig Embryos.

DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo.

Intraoperative assessment of surgical margins using "en face" frozen sections in the management of cutaneous carcinomas.

BACKGROUND: Basal cell and squamous cell carcinomas (BCC and SCC) are the most common types of cancer worldwide. Intraoperative assessment of surgical margins by frozen section has been widely used to ensure disease-free margins. The intraoperative "en face" freezing technique evaluates all peripheral and deep margins. OBJECTIVE: To report the results of the "en face" freezing technique in relation to tumor recurrence and agreement with paraffin-embedded tissue examination. METHODS: Retrospective analysis of patients undergoing surgical excision of BCC and SCC at the A. C. Camargo Cancer Center, Brazil. RESULTS: This study included 542 skin carcinomas, which were excised from 397 patients. A total of 201 male patients (50.6%), and 196 female patients (49.4%) were assessed, whose mean age was 64 years. The tumors were mostly located on the head and neck region (87.8%). BCC corresponded to 79.7% of the cases. The mean follow-up was 38 months. Tumor relapse occurred in 0.86% of the primary tumors and 3.7% of recurrent tumors. The result of the intraoperative "en face" frozen section evaluation was in agreement with the final result of the anatomopathological examination (paraffin test) in 98% of the lesions. STUDY LIMITATIONS: Not having a minimum follow-up time of 5 years for all patients. CONCLUSION: The "en face" freezing technique shows low tumor relapse, being reliable and safe to guarantee negative surgical margins of the tumor.

Enhancement of Chromatin and Epigenetic Reprogramming in Porcine SCNT Embryos-Progresses and Perspectives.

Over the last 25 years, cloned animals have been produced by transferring somatic cell nuclei into enucleated oocytes (SCNT) in more than 20 mammalian species. Among domestic animals, pigs are likely the leading species in the number of clones produced by SCNT. The greater interest in pig cloning has two main reasons, its relevance for food production and as its use as a suitable model in biomedical applications. Recognized progress in animal cloning has been attained over time, but the overall efficiency of SCNT in pigs remains very low, based on the rate of healthy, live born piglets following embryo transfer. Accumulating evidence from studies in mice and other species indicate that new strategies for promoting chromatin and epigenetic reprogramming may represent the beginning of a new era for pig cloning.

Unique SLC12A2-ROS1 fusion is associated with marked response to crizotinib in lung adenocarcinoma.

Chromosomal rearrangements involving the c-ros oncogene 1 (ROS1) gene define a subset of non-small cell lung cancers highly sensitive to small-molecule tyrosine kinase inhibitors. However, little is known about the impact of different fusion partners on tyrosine kinase inhibitor efficacy. We herein describe a case of a 26-year-old never-smoker patient from southern Africa with metastatic lung adenocarcinoma driven by SLC12A2-ROS1 fusion, who had a pronounced and durable response to crizotinib. The present case underscores the importance of pursuing actionable alterations in patients with similar clinical and epidemiological characteristics. In addition, provides the second report of crizotinib activity against lung malignancies harboring the unique SLC12A2-ROS1 fusion and highlights the importance of a deeper understanding of molecular alterations in underrepresented subgroups of patients to tailor the decision-making in daily practice.

The influence of prorenin/(pro)renin receptor on progesterone secretion by the bovine corpus luteum.

The aim of this study was to evaluate the role of prorenin/(pro)renin receptor activation on luteal progesterone (P4) secretion. Our hypothesis was that the nonproteolytic activation of (pro)renin receptor [P(RR)] is part of the regulatory mechanism responsible for corpus luteum (CL) function. In the first three experiments, prorenin was found to stimulate the production of P4, which is not inhibited by an angiotensin receptor antagonist (saralasin), but rather by a renin/prorenin inhibitor (aliskiren), a MAPK1/3 inhibitor (PD325901) or an EGFR inhibitor (AG1478), which are evidence of nonproteolytic activation of prorenin. Moreover, prorenin induced phosphorylation of MAPK1/3 in luteal cells. Following these in vitro experiments, a sequence of in vivo experiments was performed demonstrating that the intrafollicular injection of aliskiren in preovulatory follicles impaired P4 secretion in cows that ovulated. Furthermore, all profibrotic genes studied were present in the CL and TGFB1 and FN1 mRNA were upregulated from day 5-10 post-ovulation. During luteolysis, REN was downregulated at 48 h, whereas TGFB1 and SERPINE1 were dramatically upregulated in luteal tissue at 12 h after PGF. In summary, these data are evidence that nonproteolytic activation of (P)RR is involved in luteal function.

Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos.

CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14-16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts.

Heterorhabditis amazonensis to Control Euschistus heros (Hemiptera: Pentatomidae) in Laboratory and Field Conditions.

This study evaluated the potential of entomopathogenic nematodes (EPNs) to control Euschistus heros (Fabricius) under laboratory and field conditions. Eight isolates of the species Heterorhabditis amazonensis were evaluated on field-collected and laboratory-reared insect populations. The isolates that performed best in the virulence test were evaluated for in vivo production on Galleria mellonella Fabricius larvae. Then, these were evaluated in field conditions on E. heros from the laboratory and field populations. The isolates IBCB-n46, NEPET11, and IBCB-n40 caused the highest mortality in adult E. heros from laboratory rearing (100, 94, and 80%, respectively), and IBCB-n46 and NEPET11 were the best against the field population (71 and 47% mortality, respectively). In the assay to evaluate production, the isolate IBCB-n46 achieved the best result (3.4 × 105 IJs/g per larvae), followed by isolates UEPN05 (1.4 × 105 IJs/g per larvae) finally NEPET11 (7.5 × 104 IJs/g per larvae). In the assay in field conditions, all treatments differed from the control treatment, no significant differences were observed in the evaluated factors (isolates and insect populations), and the highest percentage of mortality was obtained by the UEPN05 isolate when applied to insects from the laboratory (18.57% mortality).

Hotspot analysis by confocal microscopy can help to differentiate challenging melanocytic skin lesions.

Some melanocytic lesions do not present enough clinical and dermoscopic features to allow ruling out a possible melanoma diagnosis. These "doubtful melanocytic lesions" pose a very common and challenging scenario in clinical practice and were selected at this study for reflectance confocal microscopy evaluation and subsequent surgical excision for histopathological diagnosis. The study included 110 lesions and three confocal features were statistically able to distinguish benign melanocytic lesions from melanomas: "peripheral hotspot at dermo-epidermal junction", "nucleated roundish cells at the dermo-epidermal junction" and "sheet of cells". The finding of a peripheral hotspot (atypical cells in 1mm2) at the DEJ is highlighted because has not been previously reported in the literature as a confocal feature related to melanomas.