Supplementary Material for: The “Golden Age” of Probiotics: A Systematic Review and Meta-Analysis of Randomized and Observational Studies in Preterm Infants

<p><b><i>Background:</i></b> Over the last few years, probiotics have been one of the most studied interventions in neonatal medicine. <b><i>Objectives:</i></b> The aim of this work was to analyse all studies (randomized controlled trials, RCTs, and observational studies) assessing the use of probiotics in very low birth weight (VLBW) preterm infants. <b><i>Search Methods:</i></b> A systematic literature search was conducted using PubMed, Embase, Cochrane Library, and Web of Science. The data from RCTs and observational studies were pooled and analysed separately. <b><i>Selection Criteria:</i></b> RCTs and observational studies that enrolled VLBW infants with enteral administration of probiotics were considered. Extracted study data included probiotic characteristics and at least 1 clinical outcome (necrotizing enterocolitis [NEC], late-onset sepsis or all-cause mortality). <b><i>Data Collection and Analysis:</i></b> Forty-four studies were eligible for our review: 30 RCTs and 14 observational studies. Severe NEC rates (stage II or more) and all-cause mortality were reduced among the probiotic groups in both the RCTs (RR 0.57, 95% CI 0.47-0.70, and RR 0.77, 95% CI 0.65-0.92, respectively) and the observational studies (RR 0.51, 95% CI 0.37-0.70, and RR 0.71, 95% CI 0.62-0.81, respectively). Furthermore, there was a 12% reduction in the risk of sepsis in RCTs and a 19% reduction in observational studies. The meta-analysis of observational studies showed a reduction in the risk of NEC in extremely low birth weight infants. However, this was not statistically significant. <b><i>Conclusions:</i></b> This meta-analysis of RCT and observational studies found that the use of probiotics was beneficial for the prevention of severe NEC, late-onset sepsis, and all-cause mortality in VLBW infants.</p

Supplementary Material for: Adenosine Triphosphate-Induced Rabbit Corneal Endothelial Cell Proliferation in vitro via the P2Y2-PI3K/Akt Signaling Axis

<b><i>Purpose:</i></b> To investigate the effect of the ATP-P2Y2-PI3K/Akt signaling axis on promoting rabbit corneal endothelial cell (RCEC) proliferation in vitro. <b><i>Methods:</i></b> Five concentrations of adenosine triphosphate (ATP; 1, 10, 25, 50 and 100 μM) were added to RCECs, and the cell proliferation was detected using Cell Counting Kit-8 (CCK8) and Ki67 immunohistochemical staining. Other P2Y2 receptor agonists and antagonists were added to the cells, and the proliferation effect was evaluated using CCK8 to determine the involvement of the P2Y2 receptor. Changes in the expression of phosphorylated Akt in RCECs treated with different concentrations of extracellular ATP and the duration of extracellular ATP on Akt phosphorylation were investigated using Western blotting. The pharmacological profiles with or without the PI3K/Akt pathway inhibitors were also determined using Western blotting. <b><i>Results:</i></b> We found that 10 μM ATP strongly promoted RCEC proliferation in vitro. Additionally, 25 μM ATP had a proliferation effect, whereas other concentrations (1, 50 and 100 μM) had no effect compared with the control group. Selective P2Y2 receptor agonists (UTP, ATPγS and Ap₄A) showed the same promotion effect, while P2Y2 antagonists and PI3K/Akt inhibitors inhibited the effect of ATP. Moreover, phosphorylated Akt could be induced by the addition of extracellular ATP at all five concentrations and lasted for 1 h. This phosphorylation was prevented by PI3K/Akt inhibitors and a P2Y2 antagonist. <b><i>Conclusions:</i></b> These findings showed that 10 μM ATP markedly promoted RCEC proliferation via the P2Y2-PI3K/Akt signaling axis

Supplementary Material for: LOC101930370/MiR-1471 Axis Modulates the Hedgehog Signaling Pathway in Breast Cancer

<b><i>Background/Aims:</i></b> Non-coding RNAs (ncRNAs) play vital regulatory roles in many tumors. However, the functional roles of these transcripts responsible for their dysregulation in breast cancer (BC) are not thoroughly understood. <b><i>Methods:</i></b> We examined the expression of microRNA miR-1471 in BC specimens. Online analysis tools predicted that lncRNA LOC101930370 might act as an endogenous ‘sponge’ by competing for miR-1471 binding targets. Luciferase assays were used to prove the interaction of LOC101930370, miR-1471 and SHH. Edu, wound-healing and transwell assays were used to verify the contribution of miR-1471 and LOC101930370 on MCF-7 cells proliferation and metastasis. Gain and loss of function studies were performed to evaluate the relevance of Hedgehog pathway with LOC101930370/miR-1471 regulating axis in MCF-7 cells. <b><i>Results:</i></b> The expression of miR-1471 was markedly downregulated in BC. Inhibition of miR-1471 by LOC101930370 was proved by luciferase assay. Knockdown of LOC101930370 suppressed BC cells progression. MiR-1471 inhibitor resulted in a more aggressive metastasis of MCF-7 cells. Moreover, SHH and Gli-1 expression were significantly suppressed by LOC101930370 knockdown, and upregulated by miR-1471 inhibitor transfection. <b><i>Conclusions:</i></b> Collectively, our study reveals the interaction between LOC101930370 and miR-1471 for the first time. LOC101930370 positively regulates the expression of SHH by sponging miR-1471, which sheds new light on lncRNA-directed diagnostics and therapeutics in BC

Supplementary Material for: Whole-Genome Linkage Analysis with Whole-Exome Sequencing Identifies a Novel Frameshift Variant in <b><i>NEFH</i></b> in a Chinese Family with Charcot-Marie-Tooth 2: A Novel Variant in <b><i>NEFH</i></b> for Charcot-Marie-Tooth 2

<b><i>Background:</i></b> Charcot-Marie-Tooth disease (CMT) is the most common neurodegenerative disorder of the peripheral nervous system. More than 50 genes/loci were found associated with the disease. We found a family with autosomal-dominant CMT2. <b><i>Objective:</i></b> To reveal the pathogenic gene of the family and further investigate the function of the variant. <b><i>Methods:</i></b> DNA underwent whole-genome linkage analysis for all family members and whole-exome sequencing for 2 affected members. Neurofilament light polypeptide and wild-type or mutant neurofilament heavy polypeptide (NEFH) were co-transfected into SW13 (vim–) cells. The <i>nefh</i>-knockdown zebrafish model was produced by using morpholino antisense oligonucleotides. <b><i>Results:</i></b> We identified a novel insertion variant (c.3057insG) in <i>NEFH</i> in the family. The variant led to the loss of a stop codon and an extended 41 amino acids in the protein. Immunofluorescence results revealed that mutant <i>NEFH</i> disrupted the neurofilament network and induced aggregation of NEFH protein. Knockdown of<i> nefh</i> in zebrafish caused a slightly or severely curled tail. The motor ability of <i>nefh</i>-knockdown embryos was impaired or even absent, and the embryos showed developmental defects of axons in motor neurons. The abnormal phenotype and axonal developmental defects could be rescued by injection of human wild-type but not human mutant<i> NEFH</i> mRNA. <b><i>Conclusions:</i></b> We identified a novel stop loss variant in <i>NEFH</i> that is likely pathogenic for CMT2, and the results provide further evidence for the role of an aberrant assembly of neurofilament in CMT