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MicroRNAs (miRNAs) are short, nonprotein‐coding RNAs, and their expression is dysregulated in malignant germ cell tumors (GCTs). Here, we investigated the causes and consequences of downregulated miR‐99a‐5p/miR‐100‐5p (functionally identical) and miR‐125b‐5p levels in malignant GCTs regardless of age, site, or subtype. Quantitative RT‐PCR was used to assess miR‐99a‐5p/miR‐100‐5p, miR‐125b‐5p, and associated gene expression in malignant GCT tissues/cell lines [seminoma (Sem), yolk sac tumor (YST), embryonal carcinoma (EC)]. Cells were treated with demethylating 5‐azacytidine and pyrosequencing was performed. Combination miR‐100‐5p/miR‐125b‐5p mimic replenishment was used to treat malignant GCT cells. Global messenger RNA (mRNA) targets of the replenished miRNAs were identified and Metascape used to study pathway effects. We found that expression levels of miR‐99a‐5p/miR‐100‐5p and miR‐125b‐5p, their respective pri‐miRNAs, and associated genes from chromosomes 11 and 21 (chr11/chr21) were downregulated and highly correlated in malignant GCT cells. Treatment with 5‐azacytidine caused upregulation of these miRNAs, with pyrosequencing revealing hypermethylation of their chr11/chr21 loci, likely contributing to miR‐100‐5p/miR‐125b‐5p downregulation. Combination miR‐100‐5p/miR‐125b‐5p mimic replenishment resulted in growth inhibition in Sem/YST cells, with miR‐100‐5p/miR‐125b‐5p mRNA targets enriched in downregulated genes, which were involved in cell cycle (confirmed by flow cytometry) and signaling pathways. Knockdown of the miR‐100‐5p/miR‐125b‐5p target tripartite motif containing 71 (TRIM71kd) recapitulated miR‐100‐5p/miR‐125b‐5p replenishment, with growth inhibition and cell cycle disruption of Sem/YST/EC cells. Further, replenishment led to reduced lin‐28 homolog A (LIN28A) levels and concomitant increases in let‐7 (MIRLET7B) tumor suppressor miRNAs, creating a sustained reversion of cell phenotype. In summary, combination miR‐100‐5p/miR‐125b‐5p mimic replenishment or TRIM71kd caused growth inhibition in malignant GCT cells via cell cycle disruption. Further studies are now warranted, including mimic treatment alongside conventional platinum‐based chemotherapy. MiR‐99a‐5p/miR‐100‐5p (functionally identical) and miR‐125b‐5p microRNAs are downregulated in malignant germ cell tumors (GCTs). Combination replenishment of these microRNAs using mimics resulted in growth inhibition in representative cell lines, with consequent downregulation of target genes involved in cell cycle (confirmed by flow cytometry) and signaling pathways. Further studies are now warranted, including mimic treatment alongside conventional platinum‐based chemotherapy.

BackgroundMiR-371~373 and miR-302/367 cluster over-expression occurs in all malignant germ cell tumours (GCTs), regardless of age (paediatric/adult), site (gonadal/extragonadal), or subtype [seminoma, yolk sac tumour (YST), embryonal carcinoma (EC)]. Six of eight microRNAs from these clusters contain the seed sequence ‘AAGUGC’, determining mRNA targeting. Here we sought to identify the significance of these observations by targeting these microRNAs functionally.MethodsWe targeted miR-371~373 and/or miR-302/367 clusters in malignant GCT cell lines, using CRISPR-Cas9, gapmer primary miR-302/367 transcript inhibition, and peptide nucleic acid (PNA) or locked nucleic acid (LNA)-DNA inhibition targeting miR-302a-d-3p, and undertook relevant functional assays.ResultsMiR-302/367 cluster microRNAs made the largest contribution to AAGUGC seed abundance in malignant GCT cells, regardless of subtype (seminoma/YST/EC). Following the unsuccessful use of CRISPR-Cas9, gapmer, and PNA systems, LNA-DNA-based targeting resulted in growth inhibition in seminoma and YST cells. This was associated with the de-repression of multiple mRNAs targeted by AAGUGC seed-containing microRNAs, with pathway analysis confirming predominant disruption of Rho-GTPase signalling, vesicle organisation/transport, and cell cycle regulation, findings corroborated in clinical samples. Further LNA-DNA inhibitor studies confirmed direct cell cycle effects, with an increase of cells in G0/G1-phase and a decrease in S-phase.ConclusionTargeting of specific miR-371~373 and miR-302/367 microRNAs in malignant GCTs demonstrated their functional significance, with growth inhibition mediated through cell cycle disruption.

Clinical whole-genome sequencing (WGS) has been shown to deliver potential benefits to children with cancer and to alter treatment in high-risk patient groups. It remains unknown whether offering WGS to every child with suspected cancer can change patient management. We collected WGS variant calls and clinical and diagnostic information from 281 children (282 tumors) across two English units ( n  = 152 from a hematology center, n  = 130 from a solid tumor center) where WGS had become a routine test. Our key finding was that variants uniquely attributable to WGS changed the management in ~7% (20 out of 282) of cases while providing additional disease-relevant findings, beyond standard-of-care molecular tests, in 108 instances for 83 (29%) cases. Furthermore, WGS faithfully reproduced every standard-of-care molecular test ( n  = 738) and revealed several previously unknown genomic features of childhood tumors. We show that WGS can be delivered as part of routine clinical care to children with suspected cancer and can change clinical management by delivering unexpected genomic insights. Our experience portrays WGS as a clinically impactful assay for routine practice, providing opportunities for assay consolidation and for delivery of molecularly informed patient care. In a cohort of 281 children with diagnosed or suspected cancer presenting to the NHS, implementing routine whole-genome sequencing provided clinical benefit in 29% of cases and led to change in management in 7% of patients.

MicroRNAs (miRNAs) are short, nonprotein‐coding RNAs, and their expression is dysregulated in malignant germ cell tumors (GCTs). Here, we investigated the causes and consequences of downregulated miR‐99a‐5p/miR‐100‐5p (functionally identical) and miR‐125b‐5p levels in malignant GCTs regardless of age, site, or subtype. Quantitative RT‐PCR was used to assess miR‐99a‐5p/miR‐100‐5p, miR‐125b‐5p, and associated gene expression in malignant GCT tissues/cell lines [seminoma (Sem), yolk sac tumor (YST), embryonal carcinoma (EC)]. Cells were treated with demethylating 5‐azacytidine and pyrosequencing was performed. Combination miR‐100‐5p/miR‐125b‐5p mimic replenishment was used to treat malignant GCT cells. Global messenger RNA (mRNA) targets of the replenished miRNAs were identified and Metascape used to study pathway effects. We found that expression levels of miR‐99a‐5p/miR‐100‐5p and miR‐125b‐5p, their respective pri‐miRNAs, and associated genes from chromosomes 11 and 21 (chr11/chr21) were downregulated and highly correlated in malignant GCT cells. Treatment with 5‐azacytidine caused upregulation of these miRNAs, with pyrosequencing revealing hypermethylation of their chr11/chr21 loci, likely contributing to miR‐100‐5p/miR‐125b‐5p downregulation. Combination miR‐100‐5p/miR‐125b‐5p mimic replenishment resulted in growth inhibition in Sem/YST cells, with miR‐100‐5p/miR‐125b‐5p mRNA targets enriched in downregulated genes, which were involved in cell cycle (confirmed by flow cytometry) and signaling pathways. Knockdown of the miR‐100‐5p/miR‐125b‐5p target tripartite motif containing 71 ( TRIM71 kd) recapitulated miR‐100‐5p/miR‐125b‐5p replenishment, with growth inhibition and cell cycle disruption of Sem/YST/EC cells. Further, replenishment led to reduced lin‐28 homolog A ( LIN28A ) levels and concomitant increases in let‐7 ( MIRLET7B ) tumor suppressor miRNAs, creating a sustained reversion of cell phenotype. In summary, combination miR‐100‐5p/miR‐125b‐5p mimic replenishment or TRIM71 kd caused growth inhibition in malignant GCT cells via cell cycle disruption. Further studies are now warranted, including mimic treatment alongside conventional platinum‐based chemotherapy.

Abstract Context Cardiovascular disease (CVD) is a major cause of mortality in adults with type 1 diabetes. Objective We prospectively evaluated CVD risk factors in a large, contemporary cohort of adults with type 1 diabetes living in the United States. Design Observational study of CVD and CVD risk factors over a median of 5.3 years. Setting The T1D Exchange clinic network. Patients Adults (age ≥ 18 years) with type 1 diabetes and without known CVD diagnosed before or at enrollment. Main Outcome Measure Associations between CVD risk factors and incident CVD were assessed by multivariable logistic regression. Results The study included 8,727 participants (53% female, 88% non-Hispanic white, median age 33 years [interquartile ratio {IQR} = 21, 48], type 1 diabetes duration 16 years [IQR = 9, 26]). At enrollment, median HbA1c was 7.6% (66 mmol/mol) (IQR = 6.9 [52], 8.6 [70]), 33% used a statin, and 37% used blood pressure medication. Over a mean follow-up of 4.6 years, 325 (3.7%) participants developed incident CVD. Ischemic heart disease was the most common CVD event. Increasing age, body mass index, HbA1c, presence of hypertension and dyslipidemia, increasing duration of diabetes, and diabetic nephropathy were associated with increased risk for CVD. There were no significant gender differences in CVD risk. Conclusion HbA1c, hypertension, dyslipidemia and diabetic nephropathy are important risk factors for CVD in adults with type 1 diabetes. A longer follow-up is likely required to assess the impact of other traditional CVD risk factors on incident CVD in the current era.

PurposeRecommendations to soak nuts prior to consumption to reduce phytate concentrations and improve gastrointestinal tolerance have received much attention in the popular press. This is despite no supporting scientific evidence for the practice. There is also a lack of information about how soaking nuts might affect consumer acceptability. This study primarily assessed the effects of soaking almonds on consumer acceptance and secondly assessed effects on gastrointestinal tolerance.MethodsIn this 8-week randomised crossover trial, 76 participants were allocated in balanced order to receive 30 g/day of four different preparations of almonds for 12 days: whole unsoaked, whole soaked, sliced unsoaked, and sliced soaked. Ratings of overall liking, desire to consume, and likelihood of future consumption, and severity of gastrointestinal symptoms were measured daily on visual analogue scales. The phytate concentrations were measured in all four nut types using high-performance liquid chromatography.ResultsMean acceptance ratings of all nut types were above the neutral point indicating they were acceptable. However, sliced soaked almonds were rated significantly lower overall for all three acceptance scales compared to the other treatments (all P ≤ 0.003). The sliced unsoaked almonds were rated lower than both whole nut treatments (all P ≤ 0.006), while there were no significant differences between the two whole nut treatments (all P ≥ 0.511). Gastrointestinal symptoms were minimal, but flatulence was rated significantly higher for all time points combined for soaked whole nuts compared to unsoaked whole nuts (P = 0.005). Compared to the whole unsoaked nuts (mean [SD] 531 [9] mg/100 g), phytate concentration was higher for the whole soaked almonds (563 [38] mg/100 g, P = 0.016), with no evidence of a difference for the sliced soaked almonds (548 [27] mg/100 g, P = 0.197) and no difference between the soaked forms (P = 0.262).ConclusionsThis research supports previous results suggesting nuts, including different forms, are an acceptable food. They are also well tolerated gastrointestinally, but soaking does not improve gastrointestinal tolerance or acceptance as claimed in the lay literature.