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Background: Temporary feeding tubes are commonly used but may lead to complications if malpositioned. Radiographs are the gold standard for assessing tube position, but clinician concern over radiation risks may curtail their use., Objective: We describe development and use of a reduced dose feeding tube radiograph (RDFTR) targeted for evaluation of feeding tube position., Materials and Methods: Age-based abdominal radiograph was adapted to use the lowest mAs setting of 0.32 mAs with field of view between carina and iliac crests. The protocol was tested in DIGI-13 line-pair plates and anthropomorphic phantoms. Retrospective review of initial clinical use compared dose area product (DAP) for RDFTR and routine abdomen, chest, or infant chest and abdomen. Review of RDFTR reports assessed tube visibility, malpositioning, and incidental critical findings., Results: Testing through a line-pair phantom showed loss of spatial resolution from 2.2 line pairs to 0.6 line pairs but preserved visibility of feeding tube tip in RDFTR protocol. DAP comparisons across 23,789 exams showed RDFTR reduced median DAP 72-93% compared to abdomen, 55-78% compared to chest, and 76-79% compared to infant chest and abdomen (p<0.001). Review of 3286 reports showed tube was visible in 3256 (99.1%), malpositioned in airway 8 times (0.2%) and in the esophagus 74 times (2.3%). The tip was not visualized in 30 (0.9%). Pneumothorax or pneumoperitoneum was noted seven times (0.2%) but was expected or spurious in five of these cases., Conclusion: RDFTR significantly reduces radiation dose in children with temporary feeding tubes while maintaining visibility of tube tip., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Pulmonary exacerbations (PEx) in persons with cystic fibrosis (CF) are primarily related to acute or chronic inflammation associated with bacterial lung infections, which may be caused by several bacteria that activate similar bacterial genes and produce similar by-products. The goal of our study was to perform a stratified functional analysis of bacterial genes at three distinct time points in the treatment of a PEx in order to determine the role that specific airway microbiome community members may play within each clinical state (i.e., PEx, end of antibiotic treatment, and follow-up). Our secondary goal was to compare the change between clinical states with the metabolic activity of specific airway microbiome community members., Methods: This was a prospective observational study of persons with CF treated with intravenous antibiotics for PEx between 2016 and 2020 at Children's National Hospital. Demographic and clinical information as well as respiratory samples were collected at hospital admission for PEx, end of antibiotic treatment, and follow-up. Metagenomic sequencing was performed; MetaPhlAn3 and HUMANn3 were used to assign sequences to bacterial species and bacterial metabolic genes, respectively., Results: Twenty-two persons with CF, with a mean age of 14.5 (range 7-23) years, experienced 45 PEx during the study period. Two-hundred twenty-one bacterial species were identified in the respiratory samples from the study cohort. Ten bacterial species had differential gene abundance across changes in the clinical state including Staphylococcus aureus , Streptococcus salivarius , and Veillonella atypica (all padj < 0.01 and log2FoldChange > |2|). These corresponded to a differential abundance of bacterial genes, with S. aureus accounting for 81% of the genes more abundant in PEx and S. salivarius accounting for 83% of the genes more abundant in follow-up, all compared to the end of treatment. Lastly, 8,653 metabolic pathways were identified across samples, with again S. aureus and S. salivarius contributing to the differential abundance of pathways (106 in PEx vs. 66 in follow-up, respectively). V. atypica was associated with a single metabolic pathway (UDP- N -acetyl-D-glucosamine biosynthesis) increased in follow-up compared to PEx., Discussion: Taken together, these data suggest that the metabolic potential of bacterial species can provide more insight into changes across clinical states than the relative abundance of the bacteria alone., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shumyatsky, Burrell, Chaney, Sami, Koumbourlis, Freishtat, Crandall, Zemanick and Hahn.)

Traumatic brain injury (TBI) is a risk factor for the later development of neurodegenerative diseases that may have various underlying pathologies. Chronic traumatic encephalopathy (CTE) in particular is associated with repetitive mild TBI (mTBI) and is characterized pathologically by aggregation of hyperphosphorylated tau into neurofibrillary tangles (NFTs). CTE may be suspected when behavior, cognition, and/or memory deteriorate following repetitive mTBI. Exposure to blast overpressure from improvised explosive devices (IEDs) has been implicated as a potential antecedent for CTE amongst Iraq and Afghanistan Warfighters. In this study, we identified biomarker signatures in rats exposed to repetitive low-level blast that develop chronic anxiety-related traits and in human veterans exposed to IED blasts in theater with behavioral, cognitive, and/or memory complaints. Rats exposed to repetitive low-level blasts accumulated abnormal hyperphosphorylated tau in neuronal perikarya and perivascular astroglial processes. Using positron emission tomography (PET) and the [ 18 F]AV1451 (flortaucipir) tau ligand, we found that five of 10 veterans exhibited excessive retention of [ 18 F]AV1451 at the white/gray matter junction in frontal, parietal, and temporal brain regions, a typical localization of CTE tauopathy. We also observed elevated levels of neurofilament light (NfL) chain protein in the plasma of veterans displaying excess [ 18 F]AV1451 retention. These findings suggest an association linking blast injury, tauopathy, and neuronal injury. Further study is required to determine whether clinical, neuroimaging, and/or fluid biomarker signatures can improve the diagnosis of long-term neuropsychiatric sequelae of mTBI., (© 2020. The Author(s).)

Background: Airway basal cells are specialised stem cells and regenerate airway epithelium. Airway basal cells isolated from patients with COPD regenerate airway epithelium with an abnormal phenotype. We performed gene expression analysis to gain insights into the defective regenerative programme in COPD basal cells., Methods: We conducted microarray analysis and compared COPD versus normal basal cells to identify differentially regulated genes (DEGs) and the enriched biological pathways. We determined the correlation of DEGs with cell polarisation and markers of ciliated and goblet cells. HOXB2 was knocked down in 16HBE14o- cells and monitored for polarisation of cells. HOXB2 expression in the lung sections was determined by immunofluorescence., Results: Comparison of normal and COPD basal cell transcriptomic profiles highlighted downregulation of genes associated with tissue development, epithelial cell differentiation and antimicrobial humoral response. Expression of one of the tissue development genes, HOXB2 showed strong correlation with transepithelial resistance and this gene was downregulated in COPD basal cells. Knockdown of HOXB2 , abrogated polarisation of epithelial cells in normal cells. Finally, HOXB2 expression was substantially reduced in the bronchial epithelium of COPD patients., Conclusions: Defect in gene signatures involved in tissue development and epithelial differentiation were implicated in COPD basal cells. One of the tissue developmental genes, HOXB2, is substantially reduced in bronchial epithelium of COPD patients. Since HOXB2 contributes to airway epithelial cell polarisation, we speculate that reduced expression of HOXB2 in COPD may contribute to abnormal airway epithelial regeneration in COPD., Competing Interests: Conflict of interest: F. Pineau has nothing to disclose. Conflict of interest: G. Shumyatsky has nothing to disclose. Conflict of interest: N. Owuor has nothing to disclose. Conflict of interest: N. Nalamala has nothing to disclose. Conflict of interest: S. Kotnala has nothing to disclose. Conflict of interest: S. Bolla has nothing to disclose. Conflict of interest: N. Marchetti has nothing to disclose. Conflict of interest: S. Kelsen has nothing to disclose. Conflict of interest: G.J. Criner has nothing to disclose. Conflict of interest: U.S. Sajjan has nothing to disclose., (Copyright ©ERS 2020.)

The hyperpolarization-activated cation current (termed I(h), I(q), or I(f)) was recently shown to be encoded by a new family of genes, named HCN for hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective. When expressed in heterologous cells, each HCN isoform generates channels with distinct activation kinetics, mirroring the range of biophysical properties of native I(h) currents recorded in different classes of neurons. To determine whether the functional diversity of I(h) currents is attributable to different patterns of HCN gene expression, we determined the mRNA distribution across different regions of the mouse CNS of the three mouse HCN genes that are prominently expressed there (mHCN1, 2 and 4). We observe distinct patterns of distribution for each of the three genes. Whereas mHCN2 shows a widespread expression throughout the CNS, the expression of mHCN1 and mHCN4 is more limited, and generally complementary. mHCN1 is primarily expressed within neurons of the neocortex, hippocampus, and cerebellar cortex, but also in selected nuclei of the brainstem. mHCN4 is most highly expressed within neurons of the medial habenula, thalamus, and olfactory bulb, but also in distinct neuronal populations of the basal ganglia. Based on a comparison of mRNA expression with an electrophysiological characterization of native I(h) currents in hippocampal and thalamic neurons, our data support the idea that the functional heterogeneity of I(h) channels is attributable, in part, to differential isoform expression. Moreover, in some neurons, specific functional roles can be proposed for I(h) channels with defined subunit composition.

Approximately 90% of human U6 small nuclear RNA (snRNA) contains uridine cyclic phosphate (U>p) at its 3'-end (Lund, E., and Dahlberg, J. E. (1992) Science 255, 327-330). We studied the formation of U>p at the 3' end of human U6 snRNA using an in vitro system where uridylic acid residues are added from UTP precursor and U>p is formed. Analysis of U6 snRNAs with varying number of uridylic acid residues showed that each of these species contains U>p where the phosphate originated from alpha-phosphate of UTP precursor. The cyclic phosphate formation occurred on U6 snRNA in extracts where essential spliceosomal snRNAs were specifically degraded, thereby indicating that U>p formation is not coupled to pre-mRNA splicing. A subpopulation of human signal recognition particle and mitochondrial RNA processing RNAs isolated from HeLa cells also contained cyclic phosphates at their 3' ends. These data suggest that U>p in U6 snRNA is unlikely to be related to its participation in splicing of pre-mRNAs. It appears that cyclic phosphate is an intermediate product in the metabolism of these small RNAs.

Mice with a termination codon mutation in exon 2 of the cystic fibrosis (CF) gene were generated using homologous recombination in embryonic stem cells. Animals homozygous for the mutant allele display a severe intestinal phenotype similar to that previously reported for CF mutant mice. The null nature of this allele was demonstrated by the absence of detectable wild-type mRNA, by the absence of detectable CFTR in the serous gland collecting ducts of salivary tissues, and by the lack of cAMP-mediated short-circuit current responses in colonic epithelium of mutant animals.

In eukaryotic cells, the gamma-monomethylphosphate cap structure has been identified in four small RNAs, namely, U6, 7SK, B2, and plant U3 RNAs. In this study, we show that in the case of 7SK and B2, as well as in plant U3 RNAs, the 5' stem-loop followed by a short single-stranded region serves as the capping signal. We previously showed that the nucleotides 1-25 of mouse U6 snRNA, also comprised of a stem-loop followed by a short single-stranded region, function as the capping signal. These data show that capping signals in all four RNAs have common features. The length of the stem-loop among these capped RNAs varied from 20 to 108 nucleotides, with no significant variation in the capping efficiency. In addition to the capping signal, we also observed a minimum RNA length requirement of about 15-25 nucleotides following the stem-loop for efficient capping in vitro. The capping signal in plant U3 snRNA corresponds to the additional 5' stem-loop found in U3 RNAs from plants and lower eukaryotes but absent in U3 RNA from higher animals. Consistent with this observation, the human U3 RNA that lacks the additional 5' stem-loop was not a suitable substrate for capping when compared to U6 snRNA.

We studied the role of the methylphosphate cap structure in the stability and nucleocytoplasmic transport by microinjecting U6, 7SK and B2 RNAs into the Xenopus oocytes. In every case, the methylphosphate capped RNAs were 3 to 9 times more stable than the uncapped RNAs. When a methylphosphate cap structure was placed on human H1 RNA which is normally not capped, its stability was improved 2-7 fold. These data show that the methylphosphate cap enhances the stability of 7SK, B2, H1 and U6 RNAs. The methylphosphate-capped 7SK RNA was transported into the nucleus from cytoplasm, but remained in the nucleus when injected into the nucleus; in this respect, 7SK RNA exhibited properties previously shown for U6 RNA. Both U6 and 7SK RNAs with ppp on their 5' ends were transported from cytoplasm to the nucleus suggesting that the methylphosphate cap structure is not required for transport of these RNAs across the nuclear membrane.

This is an update containing small RNA sequences published during 1991. Approximately two hundred small RNA sequences are available in this and earlier compilations. The hard copy print out of this set will be available directly from us (inquiries should be addressed to R. Reddy). These files are also available on GenBank computer. Sequences from various sources covered in earlier compilations (see Reddy, R. Nucl. Acids Res. 16:r71; Reddy, R. and Gupta, S. Nucl Acids Res. 1990 Supplement, 18:2231 and 1991 Supplement, 19:2073) are not included in this update but are listed below.

We found that hydrolysates of poly(A)+ RNA from Ehrlich ascites carcinoma cells which were transcribed by RNA polymerase III contained an unusual component designated as X. It was part of B2 RNA representing a transcript of B2 retroposon, typical of rodents. The component X possesses a cap-like structure, xppp5'G, where x has a non-nucleotide structure. About half of all B2 RNAs contained this group at the 5' end. Previously, Epstein et al. (1) detected a similar structure at the 5' end of small nuclear U6 RNA. Later, Singh and Reddy (2) showed methyl to be the blocking group in the component x of U6 RNA. Besides B2 RNA, we found 5' ends containing methyl groups in 7SK RNA.

Twelve to twenty percent of newly synthesized poly(A) + RNA is transcribed by RNA polymerase III in Ehrlich ascites carcinoma and P3O1 plasmocytoma mouse tumors. Most of this RNA designated as pol IIIpoly(A) + RNA has a size of 160 to 800 nucleotides with a maximum of distribution of ca. 300 nucleotides. Pol IIIpoly(A) + RNA fraction consists of two major classes of molecules corresponding to previously described B1 RNA and B2 RNA with the ratio of 1:4 to 2:3. All B2 RNAs present in poly(A) + fraction contain a long poly(A) segments at the 3' ends. Thus, RNA polymerase III transcripts can be polyadenylated. Several transcripts that hybridize with B2 probe were also observed in poly(A)- RNA. The major components consist of 180, 160, 120 and 95 nucleotides. The 180-nucleotide B2 RNA seems to be a primary transcript from B2 repeat. We suggest that other B2 RNAs are transcribed from truncated copies of B2 element.