A YY1-dependent increase in aerobic metabolism is indispensable for intestinal organogenesis

During late gestation, villi extend into the intestinal lumen to dramatically increase the surface area of the intestinal epithelium, preparing the gut for the neonatal diet. Incomplete development of the intestine is the most common gastrointestinal complication in neonates, but the causes are unclear. We provide evidence in mice that Yin Yang 1 (Yy1) is crucial for intestinal villus development. YY1 loss in the developing endoderm had no apparent consequences until late gestation, after which the intestine differentiated poorly and exhibited severely stunted villi. Transcriptome analysis revealed that YY1 is required for mitochondrial gene expression, and ultrastructural analysis confirmed compromised mitochondrial integrity in the mutant intestine. We found increased oxidative phosphorylation gene expression at the onset of villus elongation, suggesting that aerobic respiration might function as a regulator of villus growth. Mitochondrial inhibitors blocked villus growth in a fashion similar to Yy1 loss, thus further linking oxidative phosphorylation with late-gestation intestinal development. Interestingly, we find that necrotizing enterocolitis patients also exhibit decreased expression of oxidative phosphorylation genes. Our study highlights the still unappreciated role of metabolic regulation during organogenesis, and suggests that it might contribute to neonatal gastrointestinal disorders

Identification of myeloproliferative neoplasm drug agents via predictive simulation modeling: assessing responsiveness with micro-environment derived cytokines

Previous studies have shown that the bone marrow micro-environment supports the myeloproliferative neoplasms (MPN) phenotype including via the production of cytokines that can induce resistance to frontline MPN therapies. However, the mechanisms by which this occurs are poorly understood. Moreover, the ability to rapidly identify drug agents that can act as adjuvants to existing MPN frontline therapies is virtually non-existent. Here, using a novel predictive simulation approach, we sought to determine the effect of various drug agents on MPN cell lines, both with and without the micro-environment derived inflammatory cytokines. We first created individual simulation models for two representative MPN cell lines; HEL and SET-2, based on their genomic mutation and copy number variation (CNV) data. Running computational simulations on these virtual cell line models, we identified a synergistic effect of two drug agents on cell proliferation and viability; namely, the Jak2 kinase inhibitor, G6, and the Bcl-2 inhibitor, ABT737. IL-6 did not show any impact on the cells due to the predicted lack of IL-6 signaling within these cells. Interestingly, TNFα increased the sensitivity of the single drug agents and their use in combination while IFNγ decreased the sensitivity. In summary, this study predictively identified two drug agents that reduce MPN cell viability via independent mechanisms that was prospectively validated. Moreover, their efficacy is either potentiated or inhibited, by some of the micro-environment derived cytokines. Lastly, this study has validated the use of this simulation based technology to prospectively determine such responses

Genomic clustering analysis identifies molecular subtypes of thymic epithelial tumors independent of World Health Organization histologic type

Further characterization of thymic epithelial tumors (TETs) is needed. Genomic information from 102 evaluable TETs from The Cancer Genome Atlas (TCGA) dataset and from the IU-TAB-1 cell line (type AB thymoma) underwent clustering analysis to identify molecular subtypes of TETs. Six novel molecular subtypes (TH1-TH6) of TETs from the TCGA were identified, and there was no association with WHO histologic subtype. The IU-TAB-1 cell line clustered into the TH4 molecular subtype and in vitro testing of candidate therapeutics was performed. The IU-TAB-1 cell line was noted to be resistant to everolimus (mTORC1 inhibitor) and sensitive to nelfinavir (AKT1 inhibitor) across the endpoints measured. Sensitivity to nelfinavir was due to the IU-TAB-1 cell line’s gain-of function (GOF) mutation in PIK3CA and amplification of genes observed from array comparative genomic hybridization (aCGH), including AURKA, ERBB2, KIT, PDGFRA and PDGFB, that are known upregulate AKT, while resistance to everolimus was primarily driven by upregulation of downstream signaling of KIT, PDGFRA and PDGFB in the presence of mTORC1 inhibition. We present a novel molecular classification of TETs independent of WHO histologic subtype, which may be used for preclinical validation studies of potential candidate therapeutics of interest for this rare disease

Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes.

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease

Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council

An insight into misidentification of the small-subunit ribosomal RNA (18S rRNA) gene sequences of Theileria spp. as Theileria annulata

Abstract Background There had been isolated reports of the presence of novel Theileria annulata genotypes based on the 18S rRNA gene sequence data from India, Pakistan and Saudi Arabia; but, these studies were restricted to limited field samples. Additionally, no comparative study has been conducted on all the isolates of this parasite from different countries whose sequences are available in the nucleotide databases. Therefore, we aimed to study the genetic diversity of T. annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. Out of a total of 312 gene sequences of T. annulata available in the NCBI database, only 70 nearly complete sequences (> 1527 bp) were used for multiple sequence alignment. Results The maximum likelihood tree obtained using TN93 + G + I model manifested two major clades. All the valid host-cell transforming Theileria species clustered in one clade. The T. annulata designated sequences occupying this clade clustered together, excluding two isolates (DQ287944 and EU083799), and represented the true T. annulata sequences (n = 54). DQ287944 and EU083799 exhibited close association with Theileria lestoquardi. In addition, 14 Indian sequences formed a large monophyletic group with published Theileria orientalis sequences. The broad range of sequence identity (95.8–100%) of T. annulata designated sequences indicated the presence of different Theileria spp. A closer analysis revealed the presence of three Theileria spp., namely, T. annulata, T. orientalis, and two isolates (DQ287944 and EU083799) closely related to T. lestoquardi. The true T. annulata sequences manifested 98.8–100% nucleotide identity within them. EU083799 and 14 misidentified Indian T. annulata sequences exhibited the highest similarity with T. lestoquardi (98.6–98.8%) and T. orientalis (98.0-99.9%) in comparison with the other Theileria spp. of domestic and wild ruminants. Conclusion In the course of analyzing the genetic diversity of T. annulata, we identified the nearly complete 18S rRNA gene sequences of other Theileria spp. that have not only been misidentified as T. annulata in the GenBank™, but are also published as T. annulata. Moreover, a high level of sequence conservation was noticed in the 18S rRNA gene of true T. annulata and T. orientalis sequences

Revisiting the genotypes of Theileria equi based on the V4 hypervariable region of the 18S rRNA gene

IntroductionEquine theileriosis, an economically important disease that affects horses and other equids worldwide, is caused by a tick-borne intracellular apicomplexan protozoa Theileria equi. Genotyping of T. equi based on the 18S rRNA gene revealed the presence of two, three, four or five genotypes. In previous published reports, these genotypes have been labelled either alphabetically or numerically, and there is no uniformity in naming of these genotypes. The present study was aimed to revisit the phylogeny, genetic diversity and geographical distribution of T. equi based on the nucleotide sequences of the V4 hypervariable region of the 18S rRNA gene available in the nucleotide databases.MethodsOut of 14792 nucleotide sequences of T. equi available in the GenBank™, only 736 sequences of T. equi containing the complete V4 hypervariable region of the 18S rRNA gene (>207 bp) were used in multiple sequence alignment. Subsequently, a maximum likelihood phylogenetic tree was constructed based on the Kimura 2-parameter model (K2+I).ResultsThe phylogenetic tree placed all the sequences into four distinct clades with high bootstrap values which were designated as T. equi clades/ genotypes A, B, C and D. Our results indicated that the genotype B of Nagore et al. and genotype E of Qablan et al. together formed the clade B with a high bootstrap value (95%). Furthermore, all the genotypes probably originated from clade B, which was the most dominant genotype (52.85%) followed by clades A (27.58%), and C (9.78%) and D (9.78%). Genotype C manifested a comparatively higher genetic diversity (91.0-100% identity) followed by genotypes A (93.2-99.5%), and B and D (95.7-100%). The alignment report of the consensus nucleotide sequences of the V4 hypervariable region of the 18S rRNA gene of four T. equi genotypes (A-D) revealed significant variations in one region, between nucleotide positions 113-183, and 41 molecular signatures were recognized. As far as geographical distribution is concerned, genotypes A and C exhibited far-extending geographical distribution involving 31 and 13 countries of the Asian, African, European, North American and South American continents, respectively. On the contrary, the genotypes B and D exemplified limited distribution with confinement to 21 and 12 countries of Asian, African and European continents, respectively. Interestingly, genotypes A and C have been reported from only two continents, viz., North and South America. It was observed that genotypes A and C, and B and D exhibit similar geographical distribution.DiscussionThe present study indicated the presence of only four previously described T. equi genotypes (A, B, C and D) after performing the molecular analyses of all available sequences of the complete V4 hypervariable region of the 18S rRNA gene of T. equi isolates in the GenBank™