Paediatric progressive primary tuberculosis.

A previously healthy 13-year-old Japanese girl with a BCG vaccination history and no tuberculosis (TB) exposure history presented to the hospital with mild dyspnea for 1 month and fever for 5 days. Computed tomography showed consolidation with a pleural effusion, obstructed left main bronchus with an air bronchogram, and traction bronchiectasis of the left upper lobe (Fig. 1A, B). No improvement was observed with ampicillin. Computed tomography on day 23 showed a new granular shadow in the right upper lobe (Fig. 1C). Despite a negative interferon-gamma release assay (IGRA) result, the sputum on day 55 was positive for acid-fast bacilli on a ZiehlNeelsen stain and Mycobacterium tuberculosis on polymerase chain reaction. A fourdrug antituberculous regimen was initiated and she recovered rapidly. TB exposure history, positive tuberculin skin test or IGRA, and typical imaging findings are the triad for primary TB diagnosis (Perez-Velez and Marais, 2012; Lewinsohn et al., 2017; Ahmed et al., 2020). In pediatric primary TB, consolidation may be present and can be misdiagnosed as bacterial pneumonia; however, massive consolidation is rare (GriffithRichards et al., 2007). Primary pulmonary TB should be considered in children with lung consolidation that is unresponsive to antibiotics, despite negative IGRA and TB exposure history.

Biaryl modification of the 5'-terminus of one strand of a microRNA duplex induces strand specificity.

MicroRNAs (miRNAs) are single-stranded non-coding RNAs composed of 20-23 nucleotides. They are initially transcribed in the nucleus as pri-miRNAs. After processing, one strand from the miRNA duplex (miR-5p/miR-3p duplex) is loaded onto the RNA-induced silencing complex (RISC) to produce a functional, mature miRNA that inhibits the expression of multiple target genes. In the case of some miRNAs, both strands can be equally incorporated into the RISC as single strands, and both strands can function as mature miRNAs. Thus, a technique for selective expression of miR-5p and miR-3p strands is required to identify distinct targets of miRNAs. In this Letter, we report the synthesis and properties of miRNA duplexes carrying biaryl units at the 5'-terminus of one strand. We found that incorporation of biaryl units at the 5'-terminus of one strand of miRNA duplexes induced strand specificity in these duplexes. Further, we succeeded in identifying endogenous mRNA targets for each strand of the duplex by using the biaryl-modified miRNA duplexes.

Cdx2 and the Brm-type SWI/SNF complex cooperatively regulate villin expression in gastrointestinal cells.

In our recent study showing a correlation between Brm-deficiency and undifferentiated status of gastric cancer, we found that the Brm-type SWI/SNF complex is required for villin expression. To elucidate intestinal villin regulation more precisely, we here analyzed structure and function of the promoter of human villin. About 1.1 kb upstream of the determined major transcription start site, we identified a highly conserved region (HCR-Cdx) among mammals, which contains two binding sites for Cdx. Expression analyses of 30 human gastrointestinal cell lines suggested that villin is regulated by Cdx2. Introduction of Cdx family genes into colorectal SW480 cells revealed that villin is strongly induced strongly by Cdx2, moderately by Cdx1, and marginally by Cdx4. Knockdown of Cdx2 in SW480 cells caused a clear downregulation of villin, and reporter assays showed that HCR-Cdx is crucial for Cdx2-dependent and Brm-dependent villin expression. Immunohistochemical analyses of gastric intestinal metaplasia and cancer revealed that villin and Cdx2 expression are tightly coupled. GST pull-down assays demonstrated a direct interaction between Cdx2 and several SWI/SNF subunits. Chromatin immunoprecipitation analyses showed the recruitment of Cdx2 and Brm around HCR-Cdx. From these results, we concluded that Cdx2 regulates intestinal villin expression through recruiting Brm-type SWI/SNF complex to the villin promoter.

MicroRNAs miR-199a-5p and -3p target the Brm subunit of SWI/SNF to generate a double-negative feedback loop in a variety of human cancers.

The chromatin remodeling complex SWI/SNF is an important epigenetic regulator that includes one Brm or BRG1 molecule as catalytic subunit. Brm and BRG1 do not function identically, so this complex can regulate gene expression either positively or negatively, depending on the promoter to which it is recruited. Notably, Brm attenuation due to posttranscription suppression occurs often in human tumor cells, in which this event contributes to their oncogenic potential. Here, we report that the 3'-untranslated region of Brm mRNA has two sites that are efficiently targeted by the microRNAs miR-199a-5p and -3p, revealing a novel mechanism for modulation of Brm-type SWI/SNF activity. Computational mapping of the putative promoter region of miR-199a-2 (miPPR-199a-2) has defined it as the major contributing genetic locus for miR-199a-5p and-3p production in these tumor cell lines. We validated this predicted region by direct promoter analysis to confirm that Egr1 is a strong positive regulator of the miR-199a-2 gene. Importantly, we also showed that Egr1, miR-199a-5p, and miR-199a-3p are expressed at high levels in Brm-deficient tumor cell lines but only marginally in Brm-expressing tumor cells. Finally, we also obtained evidence that Brm negatively regulates Egr1. Together, our results reveal that miR-199a and Brm form a double-negative feedback loop through Egr1, leading to the generation of these two distinct cell types during carcinogenesis. This mechanism may offer a partial explanation for why miR-199a-5p and -3p have been reported to be either upregulated or downregulated in a variety of tumors.

Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development.

PURPOSE: To better understand microRNA miR-21 function in carcinogenesis, we analyzed miR-21 expression patterns in different stages of colorectal cancer development using in situ hybridization (ISH). EXPERIMENTAL DESIGN: Locked nucleic acid (LNA)/DNA probes and a biotin-free tyramide signal amplification system were used in ISH analyses of miRNA expression. Conditions for specific detection of miR-21 were determined using human cell lines and miR-21-expressing lentiviral vectors. Expression was determined in 39 surgically excised colorectal tumors and 34 endoscopically resected colorectal polyps. RESULTS: In the surgical samples, miR-21 expression was much higher in colorectal cancers than in normal mucosa. Strong miR-21 expression was also observed in cancer-associated stromal fibroblasts, suggesting miR-21 induction by cancer-secreted cytokines. Protein expression of PDCD4, a miR-21 target, was inversely correlated with miR-21 expression, confirming that miR-21 is indeed a negative regulator of PDCD4 in vivo. In the endoscopic samples, miR-21 expression was very high in malignant adenocarcinomas but was not elevated in nontumorigenic polyps. Precancerous adenomas also frequently showed miR-21 up-regulation. CONCLUSION: Using the LNA-ISH system for miRNA detection, miR-21 was detectable in precancerous adenomas. The frequency and extent of miR-21 expression increased during the transition from precancerous colorectal adenoma to advanced carcinoma. Expression patterns of miR-21 RNA and its target, tumor suppressor protein PDCD4, were mutually exclusive. This pattern may have clinical application as a biomarker for colorectal cancer development and might be emphasized by self-reinforcing regulatory systems integrated with the miR-21 gene, which has been previously shown in cell culture.