The \u3cem\u3elet-7\u3c/em\u3e MicroRNA Family Members \u3cem\u3emir\u3c/em\u3e-48, \u3cem\u3emir\u3c/em\u3e-84, and mir-241 Function Together to Regulate Developmental Timing in \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. We find that doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the hbl-1 3′ UTR and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions

Most \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e MicroRNAs are Individually Not Essential for Development or Viability

MicroRNAs (miRNAs), a large class of short noncoding RNAs found in many plants and animals, often act to post-transcriptionally inhibit gene expression. We report the generation of deletion mutations in 87 miRNA genes in Caenorhabditis elegans, expanding the number of mutated miRNA genes to 95, or 83% of known C. elegans miRNAs. We find that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. These observations are consistent with the hypothesis that there is significant functional redundancy among miRNAs or among gene pathways regulated by miRNAs. This study represents the first comprehensive genetic analysis of miRNA function in any organism and provides a unique, permanent resource for the systematic study of miRNAs

Mutations in Conserved Residues of the C. elegans microRNA Argonaute ALG-1 Identify Separable Functions in ALG-1 miRISC Loading and Target Repression

microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition

Leziunea iatrogenică a ductului biliar comun în timpul colecistectomiei laparoscopice la copil

Autorii prezintă un caz clinic de leziune iatrogenă a ductului biliar comun in timpul colecistectomiei laparoscopice, rezolvat prin operaţie reconstructivă – hepaticojejunostomie de ansă Roux in Y cu evoluţie clinică favorabilă. Monitorizarea clinică şi ecografică la distanţă la 2 ani postoperator nu a relevat semne de stenoza a anastomozei bilio-digesitve sau angiocolită de reflux, iar pacientul rămâne în stare satisfăcătoare.The authors present a clinical case of the iatrogenic injury of the common bile duct during laparoscopic cholecystectomy, managed by reconstructive surgery – Roux en Y hepaticojejunostomy with favorable evolution. The clinical and ultrasound follow up after 2 years postoperatively revealed no signs of stenosis of the biliodigestive anastomosis or reflux cholangitis, and the child’s condition remains satisfactory

Laparoscopic management of Gallstone disease in children

Universitatea de Stat de Medicină şi Farmacie “Nicolae Testemiţanu”, Catedra Chirurgie, Ortopedie şi Anesteziologie Pediatrică, Spitalul “Cancelariei de Stat”, Chişinău, Republica Moldova, Al XIII-lea Congres al Asociației Chirurgilor „Nicolae Anestiadi” și al III-lea Congres al Societății de Endoscopie, Chirurgie miniminvazivă și Ultrasonografie ”V.M.Guțu” din Republica MoldovaScopul studiului a fost aprecierea rolului colecistectomiei laparoscopice (CL) în tratamentuul litiazei biliare la copii. Material şi metode: studiul a inclus o analiză retrospectivă a foilor de observaţie a 21 de copii cu litiază biliară supuşi CL în Centrul Naţional Ştiinţifico-practic de Chirurgie Pediatrică “Academician Natalia Gheorghiu” în perioada decembrie 2015-martie 2019. Indicii analizaţi au inclus caracteristicele demografice, evoluţia clinică, explorările hematologice, investigaţiile imagistice, tehnica operatorie, complicaţiile postoperatorii, vindecarea postoperatorie şi diagnosticul histopatologic. Rezultate: În studiu au fost incluşi 21 de copii cu litiază biliară operaţi prin CL (8 băieţi şi 13 fete). Vârsta medie a fost de 8,3 ani (3-17 ani). 20 de copii cu fost cu litiază biliară simptomatică şi 1 copil cu litiază asimptomatică, dar cu sferocitoză ereditară. La 7 copii au fost depistaţi factori etiologici de risc pentru dezvoltarea litiazei biliare, restul 14 pacienti au fost cu colelitiază idiopatică. 17 copii au fost cu calculi pigmentari şi 4 - cu calculi colesterolici. Copiii au fost supuşi CP programate. 18 copii au suferit de colecistită cronică calculoasă şi 3 copii de colecistită acută calculoasă. La un pacient cu coledocolitiază concomitentă preoperator a fost efectuată papilosfincterotomia. Timpul mediu de operaţie a fost 56,7 minute (30-90 minute). Complicaţii postoperatorii nu au fost. Durata medie de spitalizare a fost de 4,3 zile (3-6 zile). Concluzie: Colecistectomia laparoscopică este o metodă sigura şi eficientă de tratament al litiazei biliare simptomatice la copii.The aim of the study was to assess the role of laparoscopic cholecystectomy (LC) in the treatment of pediatric gallstone disease. Material and methods: the study was based on a retrospective analysis of medical records of 21 children with cholelithiasis treated by laparoscopic cholecystectomy in the “Natalia Gheorghiu” National Scientific and Practical Center of Pediatric Surgery between December, 2015 – March, 2019. The analyzed indices included demographic characteristics, clinical evolution, blood tests, imaging results, operative technique, postoperative complications, postoperative recovery and histological diagnosis. Results: 21 children with gallstone disease were included in the study (8 boys and 13 girls). The average age was 8,3 years (range 3-17 years). 20 children had symptomatic gallstones and 1 child had asymptomatic cholelithiasis, but he also had hereditary spherocytosis. In 7 children etiologic risk factors for gallstone disease were discovered, the rest of them were with idiopathic cholelithiasis. 17 children had pigmental stones and 4 children had cholesterol stones. The elective laparoscopic cholecystectomy was performed in all children. 18 patients suffered from chronic calculous cholecystitis and 3 children had acute calculous cholecystitis. In one child with concomitant choledocholithiasis the endoscopic papillosphincterotomy was preoperatively performed. The average surgery time was 56.7 minutes (range: 30-90 minutes). There were no postoperative complications. The average length of hospitalization was of 4.3 days (range: 3-6 days). Conclusion: Laparoscopic cholecystectomy is a safe and efficient method of symptomatic pediatric gallstones treatment

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation

Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides

MicroRNAs (miRNAs) function cell-intrinsically to regulate gene expression by base-pairing to complementary mRNA targets while in association with Argonaute, the effector protein of the miRNA-mediated silencing complex (miRISC). A relatively dilute population of miRNAs can be found extracellularly in body fluids such as human blood plasma and cerebrospinal fluid (CSF). The remarkable stability of circulating miRNAs in such harsh extracellular environments can be attributed to their association with protective macromolecular complexes, including extracellular vesicles (EVs), proteins such as Argonaut 2 (AGO2), or high-density lipoproteins. The precise origins and the potential biological significance of various forms of miRNA-containing extracellular complexes are poorly understood. It is also not known whether extracellular miRNAs in their native state may retain the capacity for miRISC-mediated target RNA binding. To explore the potential functionality of circulating extracellular miRNAs, we comprehensively investigated the association between circulating miRNAs and the miRISC Argonaute AGO2. Using AGO2 immunoprecipitation (IP) followed by small-RNA sequencing, we find that miRNAs in circulation are primarily associated with antibody-accessible miRISC/AGO2 complexes. Moreover, we show that circulating miRNAs can base-pair with a target mimic in a seed-based manner, and that the target-bound AGO2 can be recovered from blood plasma in an approximately 1:1 ratio with the respective miRNA. Our findings suggest that miRNAs in circulation are largely contained in functional miRISC/AGO2 complexes under normal physiological conditions. However, we find that, in human CSF, the assortment of certain extracellular miRNAs into free miRISC/AGO2 complexes can be affected by pathological conditions such as amyotrophic lateral sclerosis