Methionine Supplementation Alleviates the Germ Cell Apoptosis Increased by Maternal Caffeine Intake in a C. elegans Model.

Caffeine (1,3,7-trimethylxanthine) is a widely consumed bioactive substance worldwide. Our recent study showed that a reduction in both reproduction and yolk protein production (vitellogenesis) caused by caffeine intake were improved by vitamin B12 supplementation, which is an essential co-factor in methionine metabolism. In the current study, we investigated the role of methionine in the reproduction of caffeine-ingested animals (CIAs). We assessed the effect of methionine metabolism on CIAs and found that caffeine intake decreased both methionine levels and essential enzymes related to the methionine cycle. Furthermore, we found that the caffeine-induced impairment of methionine metabolism decreased vitellogenesis and increased germ cell apoptosis in an LIN-35/RB-dependent manner. Interestingly, the increased germ cell apoptosis was restored to normal levels by methionine supplementation in CIAs. These results indicate that methionine supplementation plays a beneficial role in germ cell health and offspring development by regulating vitellogenesis.

Germline mutational variants of Turkish ovarian cancer patients suspected of Hereditary Breast and Ovarian Cancer (HBOC) by next-generation sequencing.

Hereditary Breast and Ovarian Cancer (HBOC) syndrome is characterized by an increased risk of developing breast cancer (BC) and ovarian cancer (OC) due to inherited genetic mutations. Understanding the genetic variants associated with HBOC is crucial for identifying individuals at high risk and implementing appropriate preventive measures. The study included 630 Turkish OC patients with confirmed diagnostic criteria of The National Comprehensive Cancer Network (NCCN) concerning HBOC. Genomic DNA was extracted from peripheral blood samples, and targeted Next-generation sequencing (NGS) was performed. Bioinformatics analysis and variant interpretation were conducted to identify pathogenic variants (PVs). Our analysis revealed a spectrum of germline pathogenic variants associated with HBOC in Turkish OC patients. Notably, several pathogenic variants in BRCA1, BRCA2, and other DNA repair genes were identified. Specifically, we observed germline PVs in 130 individuals, accounting for 20.63% of the total cohort. 76 distinct PVs in genes, BRCA1 (40 PVs), BRCA2 (29 PVs), ATM (1 PV), CHEK2 (2 PVs), ERCC2 (1 PV), MUTYH (1 PV), RAD51C (1 PV), and TP53 (1PV) and also, two different PVs (i.e., c.135-2 A>G p.? in BRCA1 and c.6466_6469delTCTC in BRCA2) were detected in a 34-year-old OC patient. In conclusion, our study contributes to a better understanding of the genetic variants underlying HBOC in Turkish OC patients. These findings provide valuable insights into the genetic architecture of HBOC in the Turkish population and shed light on the potential contribution of specific germline PVs to the increased risk of OC.

Universal Germline-Genetic Testing for Breast Cancer: Implementation in a Rural Practice and Impact on Shared Decision-Making.

BACKGROUND: Whereas the National Comprehensive Cancer Network (NCCN) criteria restrict germline-genetic testing (GGT) to a subset of breast cancer (BC) patients, the American Society of Breast Surgeons recommends universal GGT. Although the yield of pathogenic germline variants (PGV) in unselected BC patients has been studied, the practicality and utility of incorporating universal GGT into routine cancer care in community and rural settings is understudied. This study reports real-world implementation of universal GGT for patients with breast cancer and genetics-informed, treatment decision-making in a rural, community practice with limited resources. METHODS: From 2019 to 2022, all patients with breast cancer at a small, rural hospital were offered GGT, using a genetics-extender model. Statistical analyses included Fisher's exact test, t-tests, and calculation of odds ratios. Significance was set at p < 0.05. RESULTS: Of 210 patients with breast cancer who were offered GGT, 192 (91.4%) underwent testing with 104 (54.2%) in-criteria (IC) and 88 (45.8%) out-of-criteria (OOC) with NCCN guidelines. Pathogenic germline variants were identified in 25 patients (13.0%), with PGV frequencies of 15 of 104 (14.4%) in IC and ten of 88 (11.4%) in OOC patients (p = 0.495). GGT informed treatment for 129 of 185 (69.7%) patients. CONCLUSIONS: Universal GGT was successfully implemented in a rural, community practice with > 90% uptake. Treatment was enhanced or de-escalated in those with and without clinically actionable PGVs, respectively. Universal GGT for patients with breast cancer is feasible within rural populations, enabling optimization of clinical care to patients' genetic profile, and may reduce unnecessary healthcare, resource utilization.

Pathogenic Germline Variants in BRCA1/2 and p53 Identified by Real-world Comprehensive Cancer Genome Profiling Tests in Asian Patients.

Cancer genome profiling (CGP) occasionally identifies pathogenic germline variants (PGV) in cancer susceptibility genes (CSG) as secondary findings. Here, we analyzed the prevalence and clinical characteristics of PGVs based on nationwide real-world data from CGP tests in Japan. We analyzed the genomic information and clinical characteristics of 23,928 patients with solid cancers who underwent either tumor-only (n = 20,189) or paired tumor-normal (n = 3,739) sequencing CGP tests between June 2019 and December 2021 using the comprehensive national database. We assigned clinical significance for all variants and highlighted the prevalence and characteristics of PGVs. Our primary analysis of the tumor-normal sequencing cohort revealed that 152 patients (4.1%) harbored PGVs in 15 CSGs. Among 783 germline variants, 113 were annotated as PGVs, 70 as benign variants, and 600 as variants of uncertain significance. The number of PGVs identified was highest in BRCA1/2, with 56, followed by TP53, with 18. PGVs were the most prevalent in ovarian and peritoneal cancers, including among cancer types common in Asia. In the tumor-only sequencing cohort, of the 5,184 pathogenic somatic variants across 26 CSGs, 784 (15.1%) were extracted according to the European Society for Medical Oncology recommendations for germline-focused tumor analysis. The prevalence of PGVs was similar to that previously reported in Europe and the United States. This is the largest analysis based on real-world tumor-normal sequencing tests in Asia. The more widespread use of the tumor-normal sequencing CGP test could be reasonable for evaluating PGVs. SIGNIFICANCE: We analyzed real-world data from over 23,000 patients in Japan, revealing 4.1% harbored PGVs, particularly in BRCA1/2 and TP53, in CSGs. It highlights the prevalence of PGVs in Asian populations and supports the broader adoption of tumor-normal sequencing CGP tests for PGV evaluation.

Essential role of Mg2+ in mouse preimplantation embryo development revealed by TRPM7 chanzyme-deficient gametes.

TRPM7 (transient receptor potential cation channel subfamily M member 7) is a chanzyme with channel and kinase domains essential for embryo development. Using gamete-specific Trpm7-null lines, we report that TRPM7-mediated Mg2+ influx is indispensable for reaching the blastocyst stage. TRPM7 is expressed dynamically from gametes to blastocysts; displays stage-specific localization on the plasma membrane, cytoplasm, and nucleus; and undergoes cleavage that produces C-terminal kinase fragments. TRPM7 underpins Mg2+ homeostasis, and excess Mg2+ but not Zn2+ or Ca2+ overcomes the arrest of Trpm7-null embryos; expressing Trpm7 mRNA restores development, but mutant versions fail or are partially rescued. Transcriptomic analyses of Trpm7-null embryos reveal an abundance of oxidative stress-pathway genes, confirmed by mitochondrial dysfunction, and a reduction in transcription factor networks essential for proliferation; Mg2+ supplementation corrects these defects. Hence, TRPM7 underpins Mg2+ homeostasis in preimplantation embryos, prevents oxidative stress, and promotes gene expression patterns necessary for developmental progression and cell-lineage specification.

The clinical utility and costs of whole-genome sequencing to detect cancer susceptibility variants-a multi-site prospective cohort study.

BACKGROUND: Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer predisposition genes and be at higher risk for developing cancer. METHODS: This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. RESULTS: Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. CONCLUSIONS: These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability.

Conditions for transplantation of primordial germ cells in the yellowtail tetra, Astyanax altiparanae.

Biotechniques, including surrogate propagation derived from primordial germ cell (PGC) transplantation, are valuable tools for the reconstitution of endangered fish species. Although promising, there are no previous studies reporting such approaches using neotropical fish species. The aim of this study was to establish germline chimeras in neotropical fish by using the yellowtail tetra Astyanax altiparanae as a model species of the order Characiformes. Germline chimeras were obtained after transplantation of PGCs cultivated under different conditions: saline medium and supplemented with DMEM, amino acids, vitamins, glutamine, pyruvate, and fetal bovine serum, and subsequently transplanted into A. altiparanae triploids and triploid hybrids from the cross between A. altiparanae (♀) and A. fasciatus (♂). The results indicate ectopic migration in host embryos after transplantation of PGCs cultivated in saline medium. However, PGCs cultivated in supplemented medium migrated to the region of the gonadal ridge in 4.5% of triploid and 19.3% in triploid hybrid. In addition, the higher expression of dnd1, ddx4 and dazl genes was found in PGCs cultivated in supplemented culture medium. This indicates that the culture medium influences the maintenance and development of the cultivated cells. The expression levels of nanos and cxcr4b (related to the differentiation and migration of PGCs) were decreased in PGCs from the supplemented culture medium, supporting the results of ectopic migration. This is the first study to report the transplantation of PGCs to obtain germline chimera in neotropical species. The establishment of micromanipulation procedures in a model neotropical species will open new insights for the conservation of endangered species.

Challenging interpretation of germline TP53 variants based on the experience of a national comprehensive cancer centre.

TP53 variant interpretation is still challenging, especially in patients with attenuated Li-Fraumeni syndrome (LFS). We investigated the prevalence of pathogenic/likely pathogenic (P/LP) variants and LFS disease in the Hungarian population of cancer patients. By testing 893 patients with multiplex or familial cancer, we identified and functionally characterized novel splice variants of TP53 helping accurate variant classification. The differences among various semi-automated interpretation platforms without manual curation highlight the importance of focused interpretation as the automatic classification systems do not apply the TP53-specific criteria. The predicted frequency of the TP53 P/LP variants in Hungary is 0.3 per million which most likely underestimates the real prevalence. The higher detection rate of disease-causing variants in patients with attenuated LFS phenotype compared to the control population (OR 12.5; p < 0.0001) may raise the potential benefit of the TP53 genetic testing as part of the hereditary cancer panels of patients with multiple or familial cancer even when they do not meet Chompret criteria. Tumours developed at an earlier age in phenotypic LFS patients compared to the attenuated LFS patients which complicates genetic counselling as currently there are no different recommendations in surveillance protocols for LFS, phenotypic LFS, and attenuated LFS patients.

Protective effect of Mucuna pruriens (L.) DC. var. pruriens seed extract on apoptotic germ cells in ethanolic male rats.

Thai Mucuna pruriens (L.) DC. var pruriens (T-MP) seed containing levodopa (L-DOPA) and antioxidant capacity has been shown to improve sexual behavior and male reproductive parameters in rats treated with ethanol (Eth). However, its protective effect on testicular apoptotic germ cells has never been reported. This study aimed to investigate the potential effects of T-MP seed extract on expressions of caspase, proliferating cell nuclear antigen (PCNA), and dopamine D2 receptor (D2R) proteins in Eth rats. Thirty-six male Wistar rats were divided into four groups (9 animals/group), including control, Eth, T-MP150+Eth, and T-MP300+Eth, respectively. Control rats received distilled water, and Eth rats received Eth (3g/kg BW; 40%v/v). The T-MP groups were treated with T-MP seed extract at a dose of 150 or 300 mg/kg before Eth administration for 56 consecutive days. The results showed that the seminiferous tubule diameter and epithelial height were significantly increased in both T-MP treated groups compared to the Eth group. Additionally, the caspase-9 and -3, and PCNA expressions were decreased, but D2R expression was markedly increased in T-MP groups. It was concluded that T-MP seed extract could protect testicular apoptosis induced by Eth via changes in caspase, PCNA, and D2R protein expressions.

Laboratory Selection in Germline Genetic Testing: Laboratory Science Matters.

There are multiple laboratories that offer germline genetic testing, and it can be difficult to discern which one to use for testing. Some laboratories have more comprehensive analysis techniques and capability, which increases the accuracy of testing. The ordering provider has a responsibility to select the appropriate laboratory with technologic capability for the needed testing, inform the laboratory of prior testing results in the patient and family so known familial variants have targeted testing, and use appropriate terminology and nomenclature when communicating information to other healthcare professionals, patients, and families. This report presents a case illustrating the potential errors that can occur when a provider selects a laboratory that lacks the capacity to detect certain pathogenic variants, such as large deletions and duplications. False-negative germline testing results lead to missed opportunities in prevention and early detection for not only the patient but often multiple family members, which may lead to psychosocial distress and late-detected cancers. This case highlights the complexities of genetic care and why management by a genetics professional can facilitate more fiscally responsible care, appropriate genetic testing, and comprehensive care for all family members at risk.

Pivotal Role of Genetic Counselors in the Uptake of Germline Genetic Testing in Non-Metastatic Breast Cancer and the Impact of Testing on Surgical Decision Making.

BACKGROUND: Genetic testing is increasingly utilized in breast cancer patients; however, testing rates remain low. We aimed to evaluate the rate of genetic testing at a tertiary academic medical center utilizing a multidisciplinary clinic model including genetic counselor. METHODS: A single-center retrospective chart review was performed on a cohort of newly diagnosed breast cancer patients from January 2018 through February 2019. Patients were reviewed for genetic screening eligibility, consultation with a genetic counselor, and test results. RESULTS: Final analysis included 426 patients. 261 (61.3%) were found to meet National Comprehensive Cancer Network guidelines for genetic testing, of which 178 patient (68.2%) underwent testing and 32 patients (12.3%) declined testing. Of the 165 not eligible for testing, 5 patients were tested. A total of 183 patients underwent testing and 116 (63.4%) had a negative result, 17 (9.3%) were positive for at least one gene mutation and 50 (27.3%) were identified to have a variant of unknown significance (VUS). There was a positive association between those patients who met with a genetic counselor and eligibility for testing (OR 31.1, 95% CI 16.0-60.5). CONCLUSIONS: Genetic testing result has become an increasingly important factor when defining optimal surgical treatment for breast cancer patients. Increasing the availability of genetic consultation for breast cancer patients can improve testing rates and patient selection.

Long-term consumption of fermented pork fat-based diets differing in calorie, fat content, and fatty acid levels mediates oxidative stress, inflammation, redox imbalance, germ cell apoptosis, disruption of steroidogenesis, and testicular dysfunction in Wistar rats.

There is a dearth of experimental evidence available as to whether the consumption of fermented pork fat (FPF) food has any harmful effects on metabolism and reproduction due to its excessive calories, high fat content, and fatty acid methyl ester (FAME) levels. We hypothesized that exposure to a FPF-diet with excessive calories, a high fat content, and high FAME levels alters testicular physiology and metabolism, leading to permanent damage to the testicular system and its function. Thirteen-week-old male rats (n = 20) were assigned to a high-calorie, high-fat diet (FPF-H, fat-60%, 23 kJ/g), a moderate-calorie, moderate-fat diet (FPF-M, fat-30%, 17.5 kJ/g), a low-calorie and low-fat diet (FPF-L, fat-15%, 14.21 kJ/g) compared to the standard diet (Control, fat-11%, 12.56 kJ/g) orally for 90 days. GC-MS analysis of the three FPF-diets showed high quantities of saturated fatty acids (SFAs) and polyunsaturated fatty acids-ω6 (PUFA-ω6) and low levels of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids-ω3 (PUFA-ω3) compared to the control diet. Consequently, the levels of serum FAMEs of the FPF-diet fed rats were significantly increased. In addition, a high level of n-6:n-3 PUFA towards PUFA-ω6 was observed in the serum of FPF-diet fed rats due to the high content of linoleic, γ-linolenic, and arachidonic acid. Long-term consumption of FPF-diets disturbed the anthropometrical, nutritional, physiological, and metabolic profiles. Furthermore, administration of FPF-diets generated metabolic syndrome (dyslipidemia, leptinemia, insulin resistance, obesity, hepato-renal disorder and function), increased the cardiovascular risk factors, and triggered serum and testis inflammatory markers (interleukin-1↑, interleukin-6↑, interleukin-10↓, leukotriene B4↑, prostaglandin↑, nitric oxide↑, myeloperoxidase↑, lactate dehydrogenase↑, and tumor necrosis factor-α↑). Activated testis oxidative stress (conjugated dienes↑, lipid hydroperoxides↑, malondialdehyde↑, protein carbonyl↑, and fragmented DNA↑) and depleted antioxidant reserve (catalase↓, superoxide dismutase↓, glutathione S-transferase↓, reduced glutathione↓, glutathione disulfide↑, and GSH:GSSG ratio↓) were observed in FPF-diet fed rats. Disrupted testis histoarchitecture, progressive deterioration of spermatogenesis, poor sperm quality and functional indices, significant alterations in the reproductive hormones (serum and testis testosterone↓, serum estradiol↑, serum luteinizing hormone↓, and follicle-stimulating hormone↑), were noted in rats fed with FPF diets than in the control diet. Severe steroidogenic impairment (steroidogenic acute regulatory protein, StAR↓; 3ß-hydroxysteroid dehydrogenase, 3ß-HSD↓; and luteinizing hormone receptor, LHR↓), deficiency in germ cells proliferation (proliferating cell nuclear antigen, PCNA↓), and abnormally enhanced testicular germ cell apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL assay↑; B-cell lymphoma-2, BCL-2↓; Bcl-2-associated X protein, BAX↑; and BAX/BCL-2 ratio↑) were remarked in the FPF-diet administered rats in comparison with the control diet. In conclusion, the long-term feeding of an FPF-diet with excessive calories, a high fat content, and high FAME levels induced oxidative stress, inflammation, and apoptosis, resulting in metabolic syndrome and hampering male reproductive system and functions. Therefore, the adoption of FPF diets correlates with irreversible changes in testis metabolism, steroidogenesis, germ cell proliferation, and apoptosis, which are related to permanent damage to the testicular system and function later in life.

Isolation of female germline stem cells from neonatal piglet ovarian tissue and differentiation into oocyte-like cells.

It has been generally accepted that the number of oocyte pool in mammalian ovaries is limited and irreversibly consumed throughout the adulthood until menopause, which has been challenged by the existence of female germline stem cells (FGSCs) and their differentiation potentials into oocytes through mitosis. However, there have been a few reports about the existence of porcine FGSCs (pFGSCs) in the neonatal piglet ovarian tissues. In this study, the pFGSCs were isolated from the one day post partum (1 dpp) piglet ovaries by a differential anchoring velocity method combined with the magnetic cell sorting (MACS) using VASA antibody. The gene expression levels and in vitro differentiation potentials of pFGSCs were subsequently analyzed. The results showed that Oct4, C-kit, Vasa, Stella, Ifitm3 and Dazl were expressed in the pFGSCs. A small portion of pFGSCs (2.81 ± 0.76%) spontaneously differentiated into oocyte-like cells (OLCs) with a mean diameter of 50 µm and gene expressions of Vasa, Ifitm3, Blimp1, Gdf9, Zp3, Dazl and Stella. Compared with that of the spontaneous differentiation system, the differentiation rates of pFGSCs into OLCs were significantly increased after the co-supplementations of porcine follicular fluid (PFF) and retinoic acid (RA). Taken together, these above results revealed the direct evidences for the existence of pFGSCs in 1 dpp piglet ovaries and the in vitro differentiation potential of pFGSCs into OLCs, benefiting future research related to the in vitro establishment of livestock FGSCs and the in vitro differentiation of pFGSCs.

Is losartan a promising agent for the treatment of type 1 diabetes-induced testicular germ cell apoptosis in rats?

BACKGROUND: Diabetes mellitus (DM) is common metabolic disease that poses a major risk to public health and fertility. Previous studies indicate that DM may cause male infertility by triggering oxidative stress and germ cell apoptosis in the testis. Due to the undesirable effects of known antidiabetic drugs, scientists have begun to investigate the use of alternative drugs to control infertility complications observed in men. In this context, present study aimed to investigate the possible antiapoptotic effect of losartan against DM-induced testicular germ cell apoptosis. METHODS AND RESULTS: Expreimental DM model was induced by intraperitoneal injection of streptozocin (STZ, 55 mg/kg) to 28 rats, which were then randomly assigned to 4 groups; 1 mL saline solution was given to DM + saline group by oral gavage, 5 mg/kg/day oral losartan was given to DM + low-dose losartan, 20 mg/kg/day oral losartan was given to DM + mid-dose losartan and, 80 mg/kg/day oral losartan was given to DM + high-dose losartan group for 4 weeks. Bax, Bcl-2 and cleaved-Caspase 3 immunoexpression, terminal-deoxynucleotidyl transferase dutp nick end labeling (TUNEL), Annexin-V and Real Time PCR analyses performed to evaluate antiapoptotic effects of losartan on diabetic rats' testis. In addition, biochemical analyzes carried out to evaluate change in oxidative stress. CONCLUSION: The results showed that losartan may have dose-related antiapoptotic effects on rats' testis via decreasing oxidative stress.

Meiocyte size is a determining factor for unreduced gamete formation in Arabidopsis thaliana.

Polyploidy, the presence of more than two sets of chromosomes within a cell, is a widespread phenomenon in plants. The main route to polyploidy is considered through the production of unreduced gametes that are formed as a consequence of meiotic defects. Nevertheless, for reasons poorly understood, the frequency of unreduced gamete formation differs substantially among different plant species. The previously identified meiotic mutant jason (jas) in Arabidopsis thaliana forms about 60% diploid (2n) pollen. JAS is required to maintain an organelle band as a physical barrier between the two meiotic spindles, preventing previously separated chromosome groups from uniting into a single cell. In this study, we characterized the jas suppressor mutant telamon (tel) that restored the production of haploid pollen in the jas background. The tel mutant did not restore the organelle band, but enlarged the size of male jas tel meiocytes, suggesting that enlarged meiocytes can bypass the requirement of the organelle band. Consistently, enlarged meiocytes generated by a tetraploid jas mutant formed reduced gametes. The results reveal that meiocyte size impacts chromosome segregation in meiosis II, suggesting an alternative way to maintain the ploidy stability in meiosis during evolution.

SAG-dihydrochloride enhanced the expression of germ cell markers in the human bone marrow- mesenchymal stem cells (BM-MSCs) through the activation of GLI-independent hedgehog signaling pathway.

Different studies indicated that the enhancing the expression of germ cell markers improved the efficiency of stem cells in the generation of germ line cells. The aim of the present study was to investigate the effect of SAG-dihydrochloride on the expression of germ cell markers in the human bone marrow-mesenchymal stem cells (BM-MSCs). For this purpose, the human BM-MSCs were cultured in the medium containing different concentrations of SAG-dihydrochloride (10, 20 and 30 µM). After RNA extraction and cDNA synthesis, the expression level of PTCH1, GLI1, PLZF, DDX4 and STRA8 genes were determined by using SYBR Green Real time PCR. The analysis of the results obtained from PTCH1 and GLI1 expression indicated that SAG-dihydrochloride had the ability to enhance the expression of germ cell markers in a Gli-independent manner. Furthermore, the significant increased expression of STRA8 was observed in the BM-MSCs treated by 10 µM SAG-dihydrochloride for 4 and 6 days (p < 0.05). There was also the up-regulation of DDX4 in the BM-MSCs following treatment with 20 µM SAG-dihydrochloride for 4 and 6 days. The obtained results suggested that treatment with SAG-dihydrochloride increased the expression of germ cell markers in the human BM-MSCs through the activation of non-canonical sonic hedgehog signaling pathway.

Exploring the Impact of Onobrychis cornuta and Veratrum lobelianum Extracts on C. elegans: Implications for MAPK Modulation, Germline Development, and Antitumor Properties.

In an era of increasing interest in the potential health benefits of medicinal foods, the need to assess their safety and potential toxicity remains a critical concern. While these natural remedies have garnered substantial attention for their therapeutic potential, a comprehensive understanding of their effects on living organisms is essential. We examined 316 herbal extracts to determine their potential nematocidal attributes in Caenorhabditis elegans. Approximately 16% of these extracts exhibited the capacity to induce diminished survival rates and larval arrest, establishing a correlation between larval arrest and overall worm viability. Certain extracts led to an unexpected increase in male nematodes, accompanied by a discernible reduction in DAPI-stained bivalent structures and perturbed meiotic advancement, thereby disrupting the conventional developmental processes. Notably, Onobrychis cornuta and Veratrum lobelianum extracts activated a DNA damage checkpoint response via the ATM/ATR and CHK-1 pathways, thus hindering germline development. Our LC-MS analysis revealed jervine in V. lobelianum and nine antitumor compounds in O. cornuta. Interestingly, linoleic acid replicated phenotypes induced by O. cornuta exposure, including an increased level of pCHK-1 foci, apoptosis, and the MAPK pathway. Mutants in the MAPK pathway mitigated the decline in worm survival, underscoring its importance in promoting worm viability. This study reveals complex interactions between herbal extracts and C. elegans processes, shedding light on potential antitumor effects and mechanisms. The findings provide insights into the complex landscape of herbal medicine's impact on a model organism, offering implications for broader applications.

Clinical Utility of Universal Germline Genetic Testing for Patients With Breast Cancer.

Importance: National Comprehensive Cancer Network guidelines currently recommend germline testing for high-risk genes in selected patients with breast cancer. The clinical utility of recommending testing all patients with breast cancer with multigene panels is currently under consideration. Objective: To examine the implications of universal testing of patients with breast cancer with respect to clinical decision-making. Design, Setting, and Participants: Patients from a previously reported cohort were assessed as in-criteria or out-of-criteria according to the 2017 guidelines and underwent testing with a multigene germline panel between 2017 to 2018. Patients were women and men aged 18 to 90 years, with a new and/or previous diagnosis of breast cancer who had not undergone either single or multigene testing. Clinicians from 20 community and academic sites documented patient clinical information and changes to clinical recommendations made according to test findings. Association between prevalence of pathogenic or likely pathogenic germline variants and previously unreported clinical features, including scores generated by the BRCAPRO statistical model, was determined. Data were analyzed from April 2020 to May 2022. Exposure: New and/or previous diagnosis of breast cancer. Main Outcomes and Measures: Disease management recommendations that were changed as a result of genetic testing results are reported. Results: Clinicians were asked to assess changes to clinical management as a result of germline genetic testing for 952 patients. Informative clinician-reported recommendations were provided for 939 (467 in-criteria and 472 out-of-criteria) of the patients with breast cancer (936 [99.7%] female; 702 [74.8%] White; mean [SD] age at initial diagnosis, 57.6 [11.5] years). One or more changes were reported for 31 of 37 (83.8%) in-criteria patients and 23 of 34 (67.6%) out-of-criteria patients with a pathogenic or likely pathogenic variant. Recommendations were changed as a result of testing results for 14 of 22 (63.6%) out-of-criteria patients who had a variant in a breast cancer predisposition gene. Clinicians considered testing beneficial for two-thirds of patients with pathogenic or likely pathogenic variants and for one-third of patients with either negative results or variants of uncertain significance. There was no difference in variant rate between patients meeting the BRCAPRO threshold (≥10%) and those who did not (P = .86, Fisher exact test). No changes to clinical recommendations were made for most patients with negative results (345 of 349 patients [98.9%]) or variants of uncertain significance (492 of 509 patients [96.7%]). Conclusions and Relevance: In this cohort study, germline genetic testing was used by clinicians to direct treatment for most out-of-criteria patients with breast cancer with pathogenic or likely pathogenic germline variants, including those with moderate-risk variants. Universal germline testing informs clinical decision-making and provides access to targeted treatments and clinical trials for all patients with breast cancer.

Two pathways of 2n gamete formation and differences in the frequencies of 2n gametes between wild species and interspecific hybrids.

KEY MESSAGE: Epidendrum produces 2n gametes with high frequency. This paper is the first to report on multiple pathways for forming 2n gametes, meiotic defeats, and pre-meiotic chromosome doubling. Unreduced 2n reproductive cells are predominantly involved in pathways that lead to polyploid plants. Although one of the most common pathways for inducing 2n gametes is through meiotic defects, a small set of isolated species alternatively generates 2n gametes from tetraploid pollen mother cells in the pre-meiotic phase. Hence, determining the mechanisms underlying 2n gamete formation is critical to improving breeding programmes and understanding plant evolution. We investigated sporads to reveal the pathway(s) accounting for the formation and frequencies of 2n gametes in wild species and interspecific hybrids in the genus Epidendrum. We investigated different types of sporads with varying frequencies, sizes, and viability in the wild species and hybrids of the genus Epidendrum. Large tetrad-estimated pre-meiotic chromosome doubling was observed in wild species. The Epidendrum is unique in that it forms 2n pollens via two pathways, namely, meiotic defects and pre-meiotic chromosome doubling. These two pathways of 2n pollen formation could influence the high diversity generation of polyploidy with different degrees of heterozygosity and genetic backgrounds in the genus Epidendrum. Therefore, these findings are proposed to influence polyploid breeding of Epidendrum via 2n pollen, helping us understand evolution and speciation via unreduced 2n gamete formation in Orchidaceae.

Satisfaction With Clinician-Led Germline Genetic Counseling in Patients With Prostate Cancer.

PURPOSE: Indications for germline testing in prostate cancer patients have expanded substantially over the past decade. With a near-universal shortage of genetic counselors and increasing demand, increased access to genetic counseling is crucial. We sought to prospectively implement and assess a clinician-led approach to genetic counseling and testing. MATERIALS AND METHODS: Patients with metastatic or localized prostate cancer meeting National Comprehensive Cancer Network® criteria for consideration of genetic testing were offered pre-test genetic counseling by their urologist or medical oncologist as part of their routine clinical care and concurrently approached for enrollment in the Germline Genetics in Prostate Cancer Study. Consented patients filled out a post-counseling survey using validated instruments to assess the quality of counseling. For patients who elected to undergo genetic testing, an additional validated questionnaire was completed following disclosure of results. The primary outcome was the proportion of patients undergoing testing, with a target >60% of patients. The secondary outcome was overall satisfaction with counseling, with a target >85% of patients. RESULTS: A total of 275 patients enrolled, and 203 patients elected to undergo genetic testing. Post-counseling surveys were obtained from 265 patients, and post-genetic testing surveys were obtained from 132 patients. Patient satisfaction was high, with 98% of patients reporting being satisfied with the overall quality of pre-test counseling, and 74% of patients elected to undergo genetic testing. CONCLUSIONS: These results support the effectiveness of clinician-led genetic counseling in prostate cancer. With clinician training, this approach can be utilized to expand access to appropriate germline genetic testing.