Bacterial Susceptibility to Ceria Nanoparticles: The Critical Role of Surrounding Molecules.

Investigating the ternary relationship among nanoparticles (NPs), their immediate molecular environment, and test organisms rather than the direct interaction between pristine NPs and test organisms has been thrust into the mainstream of nanotoxicological research. Diverging from previous work that predominantly centered on surrounding molecules affecting the toxicity of NPs by modulating their nanoproperties, this study has unveiled a novel dimension: surrounding molecules altering bacterial susceptibility to NPs, consequently impacting the outcomes of nanobio interaction. The study found that adding nitrate as the surrounding molecules could alter bacterial respiratory pathways, resulting in an enhanced reduction of ceria NPs (nanoceria) on the bacterial surfaces. This, in turn, increased the ion-specific toxicity originating from the release of Ce3+ ions at the nanobio interface. Further transcriptome analysis revealed more mechanistic details underlying the nitrate-induced changes in the bacterial energy metabolism and subsequent toxicity patterns. These findings offer a new perspective for the deconstruction of nanobio interactions and contribute to a more comprehensive understanding of NPs' environmental fate and ecotoxicity.

Particulate Standard Establishment for Absolute Quantification of Nanoparticles by LA-ICP-MS.

The development of nanotechnology has transformed many cutting-edge studies related to single-molecule analysis into nanoparticle (NP) detection with a single-NP sensitivity and ultrahigh resolution. While laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been successful in quantifying and tracking NPs, its quantitative calibration remains a major challenge due to the lack of suitable standards and the uncertain matrix effects. Herein, we frame a new approach to prepare quantitative standards via precise synthesis of NPs, nanoscale characterization, on-demand NP distribution, and deep learning-assisted NP counting. Gold NP standards were prepared to cover the mass range from sub-femtogram to picogram levels with sufficient accuracy and precision, thus establishing an unambiguous relationship between the sampled NP number in each ablation and the corresponding mass spectral signal. Our strategy facilitated for the first time the study of the factors affecting particulate sample capture and signal transductions in LA-ICP-MS analysis and culminated in the development of an LA-ICP-MS-based method for absolute NP quantification with single-NP sensitivity and single-cell quantification capability. The achievements would herald the emergence of new frontiers cut across a spectrum of toxicological and diagnostic issues related to NP quantification.

Effects of CeO2 Nanoparticles on Nutritional Quality of Two Crop Plants, Corn (Zea mays L.) and Soybean (Glycine max L.).

With the widespread applications of manufactured nanoparticles (NPs), there are increasing concerns about their potential adverse effects on the environment and living systems. Many studies demonstrated that NPs could significantly affect the growth and development of crop plants. However, knowledge regarding the impacts of NPs on crop quality is rather limited. In this study, the effects of CeO2 NPs (25, 75, and 225 mg Ce/kg) and CeCl3 (25 mg Ce/kg) on the nutritional components of soil-cultivated corn and soybean plants were evaluated. Both treatments tended to decrease the dry weight of grain per plant, while only 225 mg/kg CeO2 NPs on soybean and CeCl3 on corn showed statistical significance compared with the respective control. CeO2 NPs at 225 mg/kg significantly decreased the content of starch in the corn kernels by 18.2% but increased total phenols in soybean seeds by 18.4%. Neither CeO2 NPs nor CeCl3 significantly affected the contents of minerals in corn kernels except for Zn. However, in the case of soybean, the two treatments tended to decrease the contents of P, Zn, Mn, and Mo but increase the content of S. Overall, the results suggest that CeO2 NPs and Ce3+ ions showed similar but not identical effects on corn and soybean plants. CeO2 NPs affect the nutritional quality of crop plants in a species-dependent manner.

Transanal full-thickness pull-through approach in the treatment of anastomotic leakage after operation for Hirschsprung disease.

BACKGROUND: Anastomotic leakage (AL) is one of the most perplexing complications that can occur following a radical operation to treat Hirschsprung disease (HSCR). The purpose of this study was to document our experience with anastomotic leakage following radical HSCR surgery to enhance therapeutic effect and prognosis. METHODS: Between January 2007 and April 2021, a retrospective study was conducted on 12 children who developed anastomotic leakage following radical surgery for HSCR. Medical records were analyzed to determine the clinical manifestations, primary surgical procedures, evaluation methods, surgical plans, and outcomes of the patients. To assess postoperative bowel function, the Rintala score was used. RESULTS: The Soave procedure was used as the primary surgical method in seven cases (58.3%), the Swenson procedure was used in four cases (33.3%), and the Rehbein procedure was used in one case (8.3%). Enterostomy (10, 83.3%) and conservative treatment (2, 16.7%) were performed when anastomotic leakage was diagnosed. Two patients who directly closed stoma without redoing pull-through both accepted enterostomy within 48 h. One female with anastomotic fistula who was closed leakage or fistula in situ had to endure lifelong stoma. Other patients who underwent redo pull-through procedures had normal bowel function. Seven patients underwent a redo pull-through procedure. Three of them preferred the transanal full-thickness pull-through (FTPT) approach, while four preferred the Soave technique. Three children had mild postoperative soiling, which improved with conservative treatment. Bowel function score was 17.5 ± 1.1. CONCLUSION: Enterostomy should be performed immediately if anastomotic leakage occurs. After leakage, it is necessary to redo the pull-through procedure in an anastomotic fistula or anastomotic stricture. Transanal FTPT reconstruction is an effective method for repairing anastomoses and leakage.

Activated M1 macrophages suppress c-kit expression via TNF-α-mediated upregulation of miR-222 in Neonatal Necrotizing Enterocolitis.

BACKGROUND: Activation of intestinal macrophages is implicated in the pathogenesis of neonatal necrotizing enterocolitis (NEC), yet its precise mechanisms remain unclear. OBJECTIVE: The purpose of this study is to investigate the role of macrophages and TNF-α via an inflammatory MicroRNA in NEC. MATERIALS AND METHODS: Immunofluorescence (IF) staining of CD68, iNOS, and Arg-1 was employed to identify phenotypes of macrophage in the intestines of NEC infants and NEC mice. Expression of TNF-α, c-kit, and miR-222 was evaluated by qRT-PCR, Western blot, and immunochemical staining from the tissue samples. RESULTS: Large number of M1 macrophage infiltration was found in the NEC intestines. Expression of CD68, iNOS, and TNF-α were significantly increased, while c-kit was decreased distinctly in the NEC group. In the early phase of NEC mouse model, inhibition of M1 macrophages reduced the incidence of NEC and intestinal inflammation. We found that TNF-α upregulated the expression of miRNA-222 and inhibited the expression of c-kit. Conversely, such decrease of c-kit expression could be reversed by miR-222 antagonists. Furtherly, dual-luciferase assay confirmed that c-kit can be inhibited by miR-222 directly. CONCLUSION: Macrophages activation in NEC intestine results in an increased inflammatory response and TNF-α production, accompanied with miR-222 upregulation and c-kit suppression. Modulations of M1 macrophages, TNF-α or miR-222 may be potential therapeutic targets for NEC treatment.

Dissolution and Retention Process of CeO2 Nanoparticles in Soil with Dynamic Redox Conditions.

The time-course association of soil physicochemical properties and fate of CeO2 nanoparticles (NPs) is not well understood. This study for the first time investigated the dissolution and retention of CeO2 NPs (<25 nm) during soil short-term (6 h) and long-term (30 d) aging processes with dynamic redox conditions. Under the additional reductant-induced initial reductive condition, theoretically, up to 220‰ of Ce(IV) was temporarily reductively dissolved within 10 min, accompanied by a slow retention process (180 min) of Ce species in soil solutions. Conversely, the dissolution and slow retention of Ce species were not significant in soil solutions without added reductant. X-ray absorption near edge spectroscopy (XANES) shows that most of Ce species were present as Ce(IV) (94.0%-97.8%) in all soils after a long-term aging process. These results indicate that the soil dynamic redox conditions induced by oxidant/reductant intrinsically determined the different time-course dissolution and retention of CeO2 NPs, highlighting the occasional reductive condition in soil solution that may contribute to the migration and diffusion of Ce species. The time-course study should be also adopted to develop a comprehensive understanding of the nano-soil interactions.

Results of rectoanal manometry after a novel laparoscopic technique: laparoscope-assisted heart-shaped anastomosis for Hirschsprung's disease.

PURPOSE: The present research utilizes a mid-term follow-up study to assess the results of anorectal manometry after laparoscope-assisted heart-shaped anastomosis (LHSA) for Hirschsprung's disease (HSCR), and compares it to a more generally applied approach, the laparoscope-assisted Soave procedure (LSP). METHODS: Retrospectively, patients from January 2015 to June 2017 who received LHSA or LSP were included in this study. After surgery, anorectal manometry was performed by the outpatient department. Anal sphincter resting pressure, anal canal length, amplitude of anal contraction, and frequency of anal contraction pre- and postoperatively were recorded. Additionally, mid-term complications were also monitored. RESULTS: Preoperative manometry showed no statistically significant difference between the LHSA and LSP groups. Postoperatively, anal sphincter resting pressure was lower in the LHSA group (60.64 ± 9.33 vs. 68.84 ± 11.80 mmHg, p = 0.001). Furthermore, anal canal length of the LHSA group was shorter than that of the LSP group (1.41 ± 0.18 vs. 1.53 ± 0.25 cm, p = 0.015). Frequency of anal contraction also showed a statistically significant difference between the LHSA and LSP groups (13.53 ± 2.17 vs. 12.50 ± 2.03 per minute, p = 0.032). The complication rates showed no significant difference and were as follows: incidence of enterocolitis was 13.89% in the LHSA group and 20.45% in the LSP group, incidence of constipation was 11.11% after LHSA and 27.27% after LSP, and incidence of soiling was 13.89% after LHSA and 25.00% after LSP. CONCLUSIONS: Manometric results of this study show satisfactory outcomes after LHSA. LHSA is an advanced surgical technique to make intestinal anastomosis easy and ensure a good prognosis.

A retrospective study of the ideal operation time for preterm biliary atresia patients.

OBJECTIVE: To study the ideal Kasai portoenterostomy (KPE) time for preterm infants with biliary atresia (BA) through evaluation of the postoperative effects. METHODS: Retrospectively, 34 preterm infants with BA from 2012 to 2016 were recruited in the present study. The following three groups were established according to their chronological and corrected age at the time of KPE operation: chronological age ≤ 90 days, chronological age > 90 days and corrected age ≤ 90 days, and corrected age > 90 days. For chronological age ≤ 90 days at operation, patients were further divided into another three groups: chronological age ≤ 60 days, chronological age > 60 days and corrected age ≤ 60 days, and corrected age > 60 days. Postoperative effects were then followed up and recorded. RESULTS: First, of those patients divided according to 90-day chronological and corrected age, postoperative total bilirubin levels (TBL), direct bilirubin levels (DBL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) of the group whose chronological age was ≤ 90 days were lower than the levels of the group whose chronological age was > 90 days and corrected age ≤ 90 days (P = 0.0472, P = 0.0358, P = 0.0083, and P = 0.0491), and the group whose corrected age was > 90 days (P = 0.0383, P = 0.0392, P = 0.0043, and P = 0.0107). Second, for those patients whose chronological age was ≤ 90 days, the group whose corrected age was > 60 days showed a higher ALT level than the other two groups with chronological age ≤ 60 days (P = 0.0472) and chronological age > 60 days and corrected age ≤ 60 days (P = 0.0258). CONCLUSION: According to the present study, the ideal KPE time for preterm BA infants should meet two conditions: chronological age ≤ 90 days and corrected age ≤ 60 days. The groups with a chronological age ≤ 60 days, and chronological age > 60 days and corrected age ≤ 60 days show similar postoperative effects.

Preparation of Activated Carbon Supported Bead String Structure Nano Zero Valent Iron in a Polyethylene Glycol-Aqueous Solution and Its Efficient Treatment of Cr(VI) Wastewater.

Nanometer zero-valent iron (nZVI) has been widely used in the treatment of heavy metals such as hexavalent chromium (Cr(VI)). A novel composite of bead string-structured nZVI on modified activated carbon (nZVI-MAC) is prepared here, using polyethylene glycol as the stable dispersant rather than traditional ethanol during the loading process. The microstructure characterization shows that nZVI particles are loaded on MAC with a bead string structure in large quantity and stably due to the addition of hydroxyl functional groups on the surface by polyethylene glycol. Experiments on the treatment of Cr(VI) in wastewater show that the reaction process requires only 20 min to achieve equilibrium. The removal rate of Cr(VI) with a low concentration (80-100 mg/L) is over 99% and the maximum saturation removal capacity is up to 66 mg/g. The system converts Cr(VI) to trivalent chromium (Cr(III)) through an oxidation-reduction effect and forms an insoluble material with iron ions by coprecipitation, which is then adsorbed on the surface of the nZVI-MAC. The process conforms to the quasi-second order adsorption kinetics equation (mainly chemical adsorption process).

Intravenous immunoglobulin for the treatment of intractable cholangitis after Kasai portoenterostomy in biliary atresia patients.

OBJECTIVE: To assess the efficacy of intravenous immunoglobulin (IVIG) as add-on treatment for intractable cholangitis (IC) after Kasai portoenterostomy (KPE) in biliary atresia (BA) patients. METHODS: 113 BA patients who had one or more episodes of cholangitis after KPE were recruited in this study. According to whether response to routine conservative treatment, all patients were divided into IC group and simple cholangitis (SC) group. Meanwhile, patients with IC subdivided into IVIG group and control group according to whether application of IVIG. RESULTS: The IC group had higher serum procalcitonin (PCT) (P = 0.014), C-reactive protein (CRP) (P = 0.023), and γ-Gltamyltranspeptidase (γ-GGT) (P = 0.031) level than the SC group. The IVIG group had shorter duration of fever after treatment (P = 0.011) and length of hospital stay (P = 0.018) than the control group. The time until recurrent episode of cholangitis was significant longer in IVIG group than in control group (P = 0.019). CONCLUSIONS: IVIG as add-on treatment may be an effective treatment for the cholangitis acute episode, and we conclude by calling for more prospective studies to attest to the role of IVIG in the treatment of cholangitis.

Performance and mechanism of Cr(VI) removal by zero-valent iron loaded onto expanded graphite.

Zero-valent iron (ZVI) was loaded on expanded graphite (EG) to produce a composite material (EG-ZVI) for efficient removal of hexavalent chromium (Cr(VI)). EG and EG-ZVI were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy and Brunauer-Emmett-Teller (BET) analysis. EG-ZVI had a high specific surface area and contained sub-micron sized particles of zero-valent iron. Batch experiments were employed to evaluate the Cr(VI) removal performance. The results showed that the Cr(VI) removal rate was 98.80% for EG-ZVI, which was higher than that for both EG (10.00%) and ZVI (29.80%). Furthermore, the removal rate of Cr(VI) by EG-ZVI showed little dependence on solution pH within a pH range of 1-9. Even at pH11, a Cr(VI) removal rate of 62.44% was obtained after reaction for 1hr. EG-ZVI could enhance the removal of Cr(VI) via chemical reduction and physical adsorption, respectively. X-ray photoelectron spectroscopy (XPS) was used to analyze the mechanisms of Cr(VI) removal, which indicated that the ZVI loaded on the surface was oxidized, and the removed Cr(VI) was immobilized via the formation of Cr(III) hydroxide and Cr(III)-Fe(III) hydroxide/oxyhydroxide on the surface of EG-ZVI.

Diagnostic value of the preoperatively detected radiological transition zone in Hirschsprung's disease.

BACKGROUND AND OBJECTIVE: The cardinal diagnostic sign of congenital aganglionic megacolon, or Hirschsprung's disease (HD), is an aganglionic segment of the distal colon or rectum. To determine the surgical planning of a radiological transition zone (TZ) in HD, this study investigated the association between a radiological TZ and the bowel resection length. METHODS: A prospective observational study was conducted in children (n = 192) with suspected HD determined by radiological TZ on contrast barium enema, and who underwent pull-through operations. The bowel resection length was ≥10 cm above the proximal radiological TZ levels and confirmed by intraoperative frozen sections. In the contrast enema, the presence and level of a radiological TZ were recorded. Correlation of the TZ features with ganglion cells assessed by immunostaining of neuronal nuclei (NeuN) and the odds ratio were calculated. RESULTS: The sensitivity and specificity for diagnosing HD by the presence of a radiological TZ were 86.9 and 92.1%, respectively; Youden's index was 79.0%. The positive and negative predictive values were 91.7 and 87.6%. The kappa value indicating an association between TZ and HD was 0.776 (P < 0.05). The correlation rate between a radiological TZ and the pathological results was 88.5% in the rectosigmoid colon and 44.4% in the descending colon, and was higher in children older than 3 months (85.3%) than in infants (69.0%). CONCLUSION: A preoperatively determined radiological TZ has potential value to identify the length of resected bowel in patients with HD, and it also has a high predictive value for diagnosis of HD.

Mutation of a regulator Ask10p improves xylose isomerase activity through up-regulation of molecular chaperones in Saccharomyces cerevisiae.

Economically feasible bioconversion of lignocelluloses into fuels and chemicals is dependent on efficient utilization of all available sugars in lignocellulosic biomass, including hextose and pentose. Previously, we constructed a xylose fermenting strain of Saccharomyces cerevisiae through metabolic engineering and enhanced its xylose utilization capability through evolutionary engineering. However, the key mechanism of improved xylose utilization and xylose isomerase activity was not identified. In this study, we applied the concept of inverse metabolic engineering to identify the factors involved in improving xylose utilization. Genomic sequencing of the evolved strain with fast xylose utilization capability was performed, and mutations possibly affecting xylose utilization were screened. Further genetic analysis of these mutant genes revealed that mutations in ASK10 (both the site-directed mutation ASK10M475R as well as ASK10 deletion), a stress response regulator-encoding gene, improved growth on xylose and enhanced xylose isomerase activity. We found that mutation of Ask10p did not increase xylose isomerase activity through interacting with the xylose isomerase protein or through directly regulating the xylA gene transcription. Although ASK10 deletion increased the copy number of the plasmid and improved the transcription of xylA, the site-direct mutation ASK10M475R did not change the plasmid copy number. Interestingly, we found that both the site-directed mutation ASK10M475R and ASK10 deletion up-regulated the transcription of molecular chaperone-encoding genes HSP26, SSA1 and HSP104, thereby facilitating the protein folding of xylose isomerase and enhancing xylose isomerase activity. This study revealed the important mechanism of chaperones in xylose isomerase activity regulation, and it provides valuable insights for efficient xylose metabolic strain development.

Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization.

Efficient and specific transporters may enhance pentose uptake and metabolism by Saccharomyces cerevisiae. Eight heterologous sugar transporters were characterized in S. cerevisiae. The transporter Mgt05196p from Meyerozyma guilliermondii showed the highest xylose transport activity among them. Several key amino acid residues of Mgt05196p were suggested by structural and sequence analysis and characterized by site-directed mutagenesis. A conserved aromatic residue-rich motif (YFFYY, position 332-336) in the seventh trans-membrane span plays an important role in D-xylose transport activity. The phenyl ring of the residue at position 336 may take the function to prevent D-xylose from escaping during uptake. F432A and N360S mutations enhanced the D-xylose transport activities of Mgt05196p. Furthermore, mutant N360F specifically transported D-xylose without any glucose-inhibition, high lighting its potential application in constructing glucose-xylose co-fermentation strains for biomass refining.

TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases.

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

Identification of signaling pathways that specify a subset of migrating enteric neural crest cells at the wavefront in mouse embryos.

During enteric nervous system (ENS) development, pioneering wavefront enteric neural crest cells (ENCCs) initiate gut colonization. However, the molecular mechanisms guiding their specification and niche interaction are not fully understood. We used single-cell RNA sequencing and spatial transcriptomics to map the spatiotemporal dynamics and molecular landscape of wavefront ENCCs in mouse embryos. Our analysis shows a progressive decline in wavefront ENCC potency during migration and identifies transcription factors governing their specification and differentiation. We further delineate key signaling pathways (ephrin-Eph, Wnt-Frizzled, and Sema3a-Nrp1) utilized by wavefront ENCCs to interact with their surrounding cells. Disruptions in these pathways are observed in human Hirschsprung's disease gut tissue, linking them to ENS malformations. Additionally, we observed region-specific and cell-type-specific transcriptional changes in surrounding gut tissues upon wavefront ENCC arrival, suggesting their role in shaping the gut microenvironment. This work offers a roadmap of ENS development, with implications for understanding ENS disorders.

Effects of soil amendments on Cd and As mobility in the soil-rice system and their distribution in the grain.

The accumulation, mobilization, and distribution of toxic metal(loid)s in rice are key factors that affect food security and determine bio-utilization patterns. In this study, five soil amendments with different components were used in paddy fields to study the key factors: organic amendments: (1) polyaspartic acid (OA1) and (2) organic fertilizer (OA2); inorganic amendments: (3) kaolinite (IA1) and (4) magnesium slag (IA2); and organic-inorganic composite amendments: (5) modified biochar/quicklime (OIA). Although the Cd and As exhibited opposite chemical dissolution behaviors, IA1/OIA, can simultaneously reduce their accumulation and transfer coefficients in rice tissues, while other amendments only work for one of them. The in situ distribution in grains showed that IA1/OIA changed the original Cd distribution in the lemma and palea, whereas all amendments reduced Cd accumulation in the germ. In contrast, OA1/IA2 amendments led to more As accumulation in the rice husks and bran than in the endosperm center, and the germ had higher As signals. Because of their similar transport pathways and interactions, the concentrations of Cd and As in the grains were correlated with a variety of mineral elements (Fe, Mo, Zn, etc.). Changes in the Cd/As concentration and distribution in rice were achieved through the improvement of soil properties and plant growth behavior through amendments. The application of OIA resulted in the highest immobilization indices, at 82.17 % and 35.34 % for Cd and As, respectively. The Cd/As concentrations in the rice grains were highly positively correlated with extractable-Cd/As in the soil (Cd: R2 = 0.95, As: R2 = 0.93). These findings reveal the migration and distribution mechanisms of Cd and As in the soil-rice system, and thus provide fundamental information for minimizing food safety risk.

RNA recording in single bacterial cells using reprogrammed tracrRNAs.

Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells.

Reprogramming TracrRNAs for Multiplexed RNA Detection.

CRISPR-based detection and recording technologies are gaining increasing attention in disease surveillance and prevention. In this chapter, we describe how our recent discovery of noncanonical crRNAs inspired the engineering of reprogrammed tracrRNAs and led to a powerful platform for multiplexed RNA detection. We provide detailed protocols regarding how to design reprogrammed tracrRNA and carry out assays in vitro and in vivo.

The utility of the 24-h delayed film of barium enema for detecting the dysganglionic bowel segment in Hirschsprung's disease.

Background: Preoperative evaluation of the dysganglionic bowel segment is critical for establishing the optimal resection strategy for Hirschsprung's disease (HSCR), which facilitates patient outcomes. Objective: We set out to determine the utility of the 24-h delayed film of barium retention in predicting the length of dysganglionic bowel segment in HSCR. Materials and methods: A retrospective study of patients with clinically suspicious HSCR who underwent a preoperative 24-h delayed film of barium enema and were surgically treated from January 2015 to December 2019 was conducted. Results: Two hundred and 58 patients were enrolled in this study. The sensitivity, specificity, positive and negative predictive values (NPVs) of the 24-h delayed film of barium enema to predict the neuropathological segment were 89.1, 91.5, 91.3, and 89.4%, respectively. The Youden index was 80.6%, with a kappa value of 0.806 (P < 0.001). The correlation rate between barium retention level and pathological results was 72.7% (16/22) when aganglionosis was restricted within the mid-distal rectum (short-segment type), increasing to 92.0% (46/50) and 93.5% (174/186) for patients that had aganglionosis extended beyond the mid-distal rectum (classical type) and sigmoid colon (long-segment type), respectively. Lastly, patients younger than 3 months showed a lower correlation rate (72.2%) compared to patients aged 3-12 months (91.0%) and > 12 months (92.6%). Conclusions: Our investigation of the 24-h delayed film of barium enema performed for patients suspected of having HSCR indicated that the barium retention level remains crucial in predicting dysganglionic bowel segment, which contributes to the decision-making for surgical physicians.