Amplified Genes May Be Overexpressed, Unchanged, or Downregulated in Cervical Cancer Cell Lines

Several copy number-altered regions (CNAs) have been identified in the genome of cervical cancer, notably, amplifications of 3q and 5p. However, the contribution of copy-number alterations to cervical carcinogenesis is unresolved because genome-wide there exists a lack of correlation between copy-number alterations and gene expression. In this study, we investigated whether CNAs in the cell lines CaLo, CaSki, HeLa, and SiHa were associated with changes in gene expression. On average, 19.2% of the cell-line genomes had CNAs. However, only 2.4% comprised minimal recurrent regions (MRRs) common to all the cell lines. Whereas 3q had limited common gains (13%), 5p was entirely duplicated recurrently. Genome-wide, only 15.6% of genes located in CNAs changed gene expression; in contrast, the rate in MRRs was up to 3 times this. Chr 5p was confirmed entirely amplified by FISH; however, maximum 33.5% of the explored genes in 5p were deregulated. In 3q, this rate was 13.4%. Even in 3q26, which had 5 MRRs and 38.7% recurrently gained SNPs, the rate was only 15.1%. Interestingly, up to 19% of deregulated genes in 5p and 73% in 3q26 were downregulated, suggesting additional factors were involved in gene repression. The deregulated genes in 3q and 5p occurred in clusters, suggesting local chromatin factors may also influence gene expression. In regions amplified discontinuously, downregulated genes increased steadily as the number of amplified SNPs increased (p<0.01, Spearman's correlation). Therefore, partial gene amplification may function in silencing gene expression. Additional genes in 1q, 3q and 5p could be involved in cervical carcinogenesis, specifically in apoptosis. These include PARP1 in 1q, TNFSF10 and ECT2 in 3q and CLPTM1L, AHRR, PDCD6, and DAP in 5p. Overall, gene expression and copy-number profiles reveal factors other than gene dosage, like epigenetic or chromatin domains, may influence gene expression within the entirely amplified genome segments

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

INFLUENCE OF ESTRUS PRE-SYNCHRONIZATION ON REPRODUCTIVE VARIABLES IN KATAHDIN EWES CARRIERS OF THE GDF9 GENE

Background: Genetic improvement in the sheep species focuses on increasing the number of offspring per sheep. Heritable characteristics such as ovulation rate, fertility, and prolificacy are desirable. Objective: To evaluate the influence of estrus pre-synchronization with PGF2α on the manifestation of estrus, onset and return to estrus (1st and 2nd), pregnancy, lambing, prolificacy, and fertility in Katahdin ewes carrying exon 2 of the gene Factor Growth and Differentiation 9 (GDF9). Methodology: Seventy-two ewes were randomized into four treatments (T): T1 (n = 18), ewes without GDF9 gene and without estrus pre-synchronization; T2 (n = 17), ewes without GDF9 gene and with estrus pre-synchronization; T3 (n = 19) ewes with the GDF9 gene and without estrus pre-synchronization, and T4 (n = 18), ewes with the GDF9 gene and with estrus pre-synchronization. Results: The presence of estrus, the onset of estrus, and returns to estrus, pregnancy, and lambing percentages were not different between treatments (p>0.05). The average pregnancy and lambing in both cases were 86.2% and the average general prolificacy was 1.4 lambs per ewe for all four treatments. There were also no significant differences for the prolificacy or fertility rate (p>0.05). Implications: The present study contributes to the understanding of the use of presynchronization with PGF2α and the effect of the presence of exon 2 of the GDF9 gene on reproductive variables. Conclusions: The presynchronization of estrus with PGF2α and the presence of exon 2 of the GDF9 gene in ewes of the Katahdin breed did not have a significant effect on the reproductive variables evaluated

Isolation, biochemical characterization, and phylogeny of a cellulose-degrading ruminal bacterium

Abstract Background: The isolation of cellulolytic bacteria, which hydrolyze cellulose to cellobiose and glucose, can provide useful information about rumen diversity. Objective: To identify and characterize a microorganism capable of hydrolyzing cellulose, isolated from a cow rumen. Methods: Anaerobic culture techniques were used for isolating cellulose-degrading rumen bacteria. Congo red staining was used to evaluate β-D-glucanase activity, and carbohydrate fermentation pattern was obtained with the kit API 50CHB/E. DNA extraction was performed and the 16S rDNA gene was amplified using 8F (5’-AGA GTT TGA TCC TGG CTC AG-3’), and 1492R (5’ GGT TAC CTT GTT ACG ACT T 3’) primers. The phylogenetic tree was reconstructed with the algorithm of maximum parsimony (bootstrap 5000), and 16S rDNA sequence was deposited in the NCBI database (accession number: KM094184). Results: The isolated bacterium showed cellulolytic activity detected with Congo red; besides, glycerol, ribose, xylose, sucrose, galactose and glucose were fermented by this bacterium. However, biochemical tests did not identify the bacteria because no match was found at database of API WEB Software. The phylogenetic inference indicated that this bacterium belongs to Shigella genus, with 98% maximal identity respect to the other taxonomic species. Conclusions: Phylogenetic analysis of 16S rRNA genes showed that the rumen isolated bacterium was a member of the genus Shigella, which, under mesophilic conditions, is an interesting candidate for obtaining oligosaccharides from lignocellulosic biomass.Resumo Antecedentes: As bactérias celulolíticas hidrolizam a celulosa em celobiose e glicose, e o isolamento desses microrganismos fornece informações sobre a diversidade do rúmen. Objetivo: Identificar e caracterizar um microorganismo isolada do rúmen de uma vaca, com capacidade para hidrolisar a celulose. Métodos: Técnicas de cultura anaeróbica foram utilizadas para isolar bactérias ruminais que degradam a celulose. A atividade β-D-glucanase foi mostrada utilizando mancha de vermelho Congo, e o padrão de fermentação de carbohidratos foi obtida com o kit API 50CHB/E. A extracção foi realizada de DNA e amplificou-se os genes 16S rDNA utilizando os iniciadores 8F (AGA GTT TGA 5’-TCC TGG CTC AG-3’), e 1492R (5’ CTT GGT TAC GTT ACG TCA T 3’). A árvore filogenética foi reconstruída com o algoritmo de máxima parcimônia (réplicas 5000). A sequência de rDNA 16S foi depositada no banco de dados do NCBI (número de acesso: KM094184). Resultados: O isolado mostrou uma atividade celulolítica com coloração vermelho Congo; además esta bactéria fermentação de glicerol, ribose, xilose, sacarose, galactose e glicose. No entanto, com as provas bioquímicas não se identificou a bactérias isolada, já que não se encontrou na base de dados do software API WEB. A inferência filogenética indicou que esta bactéria pertence ao género Shigella, com 98% de identidade de máximo respeito para outras espécies taxonômicas. Conclusão: A análise filogenética do gene 16S rRNA mostrou as bactérias isoladas do ambiente ruminal como um membro do género Shigella, que condições mesofilicas é um candidato atraente para obter oligossacarídeos da biomassa lignocelulósica.Resumen Antecedentes: El aislamiento de las bacterias celulolíticas, que hidrolizan la celulosa a celobiosa y glucosa, proporciona valiosa información sobre la diversidad del rumen. Objetivo: Identificar y caracterizar un microorganismo capaz de hidrolizar celulosa, aislado de un rumen vacuno. Métodos: Se utilizaron técnicas de cultivo anaeróbico para aislar bacterias ruminales que degradan celulosa. La tinción con rojo Congo se usó para evaluar la actividad β-D-glucanasa y el patrón de fermentación de carbohidratos se obtuvo con el kit API 50CHB/E. Se realizó la extracción de DNA y se amplificó el gen de 16S rDNA utilizando los cebadores 8F (5’-AGA GTT TGA TCC TGG CTC AG-3’), y 1492R (5’ GGT TAC CTT GTT ACG ACT T 3’). El árbol filogenético se reconstruyó con el algoritmo de máxima parsimonia (replicas 5000) y la secuencia 16S rDNA se depositó en la base de datos del NCBI (número de acceso: KM094184). Resultados: La bacteria aislada mostró actividad celulolítica detectada con la tinción de rojo Congo; además, esta bacteria fermenta glicerol, ribosa, xilosa, sacarosa, galactosa y glucosa. Sin embargo, las pruebas bioquímicas no permitieron identificar a la bacteria aislada, por no encontrar coincidencias en la base de datos del software API WEB. La inferencia filogenética indicó que esta bacteria pertenece al género Shigella, con 98% de identidad máxima respecto a las otras especies taxonómicas. Conclusiones: El análisis filogenético del gen 16S rRNA mostró que la bacteria aislada del rumen es un miembro del género Shigella que, en condiciones mesófilas, es un candidato interesante para obtener oligosacáridos a partir de biomasa lignocelulósica

Mitosis Is a Source of Potential Markers for Screening and Survival and Therapeutic Targets in Cervical Cancer

<div><p>The effect of preventive human papillomavirus (HPV) vaccination on the reduction of the cervical cancer (CC) burden will not be known for 30 years. Therefore, it’s still necessary to improve the procedures for CC screening and treatment. The objective of this study was to identify and characterize cellular targets that could be considered potential markers for screening or therapeutic targets. A pyramidal strategy was used. Initially the expression of 8,638 genes was compared between 43 HPV16-positive CCs and 12 healthy cervical epitheliums using microarrays. A total of 997 genes were deregulated, and 21 genes that showed the greatest deregulation were validated using qRT-PCR. The 6 most upregulated genes (<em>CCNB2, CDC20, PRC1, SYCP2, NUSAP1</em>, <em>CDKN3</em>) belong to the mitosis pathway. They were further explored in 29 low-grade cervical intraepithelial neoplasias (CIN1) and 21 high-grade CIN (CIN2/3) to investigate whether they could differentiate CC and CIN2/3 (CIN2+) from CIN1 and controls. <em>CCNB2</em>, <em>PRC1</em>, and <em>SYCP2</em> were mostly associated with CC and <em>CDC20</em>, <em>NUSAP1</em>, and <em>CDKN3</em> were also associated with CIN2/3. The sensitivity and specificity of <em>CDKN3</em> and <em>NUSAP1</em> to detect CIN2+ was approximately 90%. The proteins encoded by all 6 genes were shown upregulated in CC by immunohistochemistry. The association of these markers with survival was investigated in 42 CC patients followed up for at least 42 months. Only <em>CDKN3</em> was associated with poor survival and it was independent from clinical stage (HR = 5.9, 95%CI = 1.4–23.8, p = 0.01). <em>CDKN3</em> and <em>NUSAP1</em> may be potential targets for the development of screening methods. Nevertheless, further studies with larger samples are needed to define the optimal sensitivity and specificity. Inhibition of mitosis is a well-known strategy to combat cancers. Therefore, <em>CDKN3</em> may be not only a screening and survival marker but a potential therapeutic target in CC. However, whether it’s indispensable for tumor growth remains to be demonstrated.</p> </div