High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity.

Recurrent and functional regulatory mutations in breast cancer.

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.

Publisher Correction: Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity.

RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

Evaluation of endocrine resistance using ESR1 genotyping of circulating tumor cells and plasma DNA.

PURPOSE: Therapeutic efficacy of hormonal therapies to target estrogen receptor (ER)-positive breast cancer is limited by the acquisition of ligand-independent ESR1 mutations, which confer treatment resistance to aromatase inhibitors (AIs). Monitoring for the emergence of such mutations may enable individualized therapy. We thus assessed CTC- and ctDNA-based detection of ESR1 mutations with the aim of evaluating non-invasive approaches for the determination of endocrine resistance. PATIENTS AND METHODS: In a prospective cohort of 55 women with hormone receptor-positive metastatic breast cancer, we isolated circulating tumor cells (CTCs) and developed a high-sensitivity method for the detection of ESR1 mutations in these CTCs. In patients with sufficient plasma for the simultaneous extraction of circulating tumor DNA (ctDNA), we performed a parallel analysis of ESR1 mutations using multiplex droplet digital PCR (ddPCR) and examined the agreement between these two platforms. Finally, we isolated single CTCs from a subset of these patients and reviewed RNA expression to explore alternate methods of evaluating endocrine responsiveness. RESULTS: High-sensitivity ESR1 sequencing from CTCs revealed mono- and oligoclonal mutations in 22% of patients. These were concordant with plasma DNA sequencing in 95% of cases. Emergence of ESR1 mutations was correlated both with time to metastatic relapse and duration of AI therapy following such recurrence. The Presence of an ESR1 mutation, compared to ESR1 wild type, was associated with markedly shorter Progression-Free Survival on AI-based therapies (p = 0.0006), but unaltered to other non-AI-based therapies (p = 0.73). Compared with ESR1 mutant cases, AI-resistant CTCs with wild-type ESR1 showed an elevated ER-coactivator RNA signature, consistent with their predicted response to second-line hormonal therapies. CONCLUSION: Blood-based serial monitoring may guide the selection of precision therapeutics for women with AI-resistant ER-positive breast cancer.

Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

[Clinical features of coronavirus disease 2019 in children aged <18 years in Jiangxi, China: an analysis of 23 cases].

OBJECTIVE: To study the clinical features of coronavirus disease 2019 (COVID-19) in children aged <18 years. METHODS: A retrospective analysis was performed from the medical data of 23 children, aged from 3 months to 17 years and 8 months, who were diagnosed with COVID-19 in Jiangxi, China from January 21 to February 29, 2020. RESULTS: Of the 23 children with COVID-19, 17 had family aggregation. Three children (13%) had asymptomatic infection, 6 (26%) had mild type, and 14 (61%) had common type. Among these 23 children, 16 (70%) had fever, 11 (48%) had cough, 8 (35%) had fever and cough, and 8 (35%) had wet rales in the lungs. The period from disease onset or the first nucleic acid-positive detection of SARS-CoV-2 to the virus nucleic acid negative conversion was 6-24 days (median 12 days). Of the 23 children, 3 had a reduction in total leukocyte count, 2 had a reduction in lymphocytes, 2 had an increase in C-reactive protein, and 2 had an increase in D-dimer. Abnormal pulmonary CT findings were observed in 12 children, among whom 9 had patchy ground-glass opacities in both lungs. All 23 children received antiviral therapy and were recovered. CONCLUSIONS: COVID-19 in children aged <18 years often occurs with family aggregation, with no specific clinical manifestation and laboratory examination results. Most of these children have mild symptoms and a good prognosis. Epidemiological history is of particular importance in the diagnosis of COVID-19 in children aged <18 years.

Crizotinib in ROS1-rearranged non-small-cell lung cancer.

BACKGROUND: Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non-small-cell lung cancers (NSCLCs) that may be susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and another proto-oncogene receptor tyrosine kinase, MET. METHODS: We enrolled 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety, pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of next-generation sequencing or reverse-transcriptase-polymerase-chain-reaction assays. RESULTS: The objective response rate was 72% (95% confidence interval [CI], 58 to 84), with 3 complete responses and 33 partial responses. The median duration of response was 17.6 months (95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4 to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No correlation was observed between the type of ROS1 rearrangement and the clinical response to crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged NSCLC. CONCLUSIONS: In this study, crizotinib showed marked antitumor activity in patients with advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which crizotinib is highly active. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

MET Exon 14 Skipping in Non-Small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancers (NSCLCs) harboring specific genetic alterations can be highly sensitive to targeted therapies. MATERIALS AND METHODS: We performed a targeted rearrangement assay on 54 NSCLCs across all stages that were from patients who were never smokers and did not have driver mutations. Because MET exon 14 skipping was the most frequent alteration found, we surveyed the results for MET exon 14 skipping at Massachusetts General Hospital (MGH) since the inclusion of this alteration into our current molecular profiling panel. RESULTS: In a cohort of 54 never-smokers with lung cancers that were wild-type for known driver mutations, MET exon 14 skipping was the most frequently recurring alteration, occurring in 10 cancers (19%). Clinical testing at MGH via our next-generation sequencing (NGS) and NGS-rearrangement panels showed an additional 16 cases of MET exon 14 skipping, for an overall estimated frequency of 5.6%. A clinical case of a patient with MET exon 14 skipping treated with the MET inhibitor crizotinib is also described. CONCLUSION: MET exon 14 skipping is a targetable gene alteration found in NSCLC. Patients with these alterations may respond well to MET inhibition. IMPLICATIONS FOR PRACTICE: MET exon 14 skipping occurs with an approximately 5% frequency in NSCLC and is seen in both squamous and adenocarcinoma histology. Patients whose cancers have MET exon 14 skipping can respond well to MET inhibitors. Molecular testing for MET exon 14 skipping should be performed on all lung cancers because this is a targetable alteration.

Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts.

BACKGROUND AND AIMS: The value of next-generation sequencing (NGS) of pancreatic cyst fluid relative to the clinical and imaging impression has not been well-studied. The aim of this study was to assess the impact of NGS on the clinical diagnosis from imaging and carcinoembryonic antigen (CEA) and thus the management of pancreatic cysts. METHODS: Ninety-two pancreatic cyst fluids from 86 patients were analyzed by cytology, CEA, and targeted NGS. Cysts were classified by imaging as nonmucinous, mucinous, or not specified. NGS results were compared with the imaging impression stratified by CEA and cytology. RESULTS: NGS impacted the clinical diagnosis by defining a cyst as mucinous in 48% of cysts without elevated CEA levels. The VHL gene in 2 intraductal papillary mucinous neoplasms (IPMNs) supported a serous cystadenoma. Twenty percent of cysts that were nonmucinous by imaging were mucinous by NGS. Of the 14 not-specific cysts, CEA levels were not elevated in 12 (86%), and NGS established a mucinous etiology in 3 (25%). A KRAS or GNAS mutation supported an IPMN with nonmucinous CEA in 71%. A KRAS mutation reclassified 19% of nonneoplastic cysts with nonmucinous CEA as mucinous. Seven cyst fluids (8%) had either a TP53 mutation or loss of CDKN2A or SMAD4 in addition to KRAS and/or GNAS mutations; 5 of 7 (71%) were clinically malignant, and high-grade cytology was detected in all 5. Overall, CEA was more specific for a mucinous etiology (100%), but NGS was more sensitive (86% vs 57%). CONCLUSIONS: NGS of pancreatic cyst fluid impacts clinical diagnosis and patient management by defining, supporting, or changing the clinical diagnosis based on imaging and CEA. NGS was most valuable in identifying mucinous cysts with nonmucinous CEA. An added benefit is the potential to detect mutations late in the progression to malignancy that may increase the risk classification of the cyst based on imaging and cytology.

Severity of Acute Cholecystitis and Risk of Iatrogenic Bile Duct Injury During Cholecystectomy, a Population-Based Case-Control Study.

BACKGROUND: Acute cholecystitis is a common complication to gallstone disease. The relation between the severity of acute cholecystitis and risk of bile duct injury during cholecystectomy has not yet been addressed and is the main focus of this study. METHODS: All cases with iatrogenic bile duct injury during cholecystectomy, within the Lake Mälaren region, Sweden, were identified through ICD procedure codes for biliary reconstruction within the Swedish Inpatient Register and matched to non-injured cholecystectomized controls. Information regarding perioperative variables was collected through medical record review. RESULTS: After review, 158 cases and 623 controls remained for analyses. Adjusted risk of bile duct injury was doubled among patients with acute cholecystitis (OR 1.97 95 % CI 1.05-3.72), whereas a mild acute cholecystitis (Tokyo grade I) did not affect the risk of bile duct injury (OR 0.96 95 % CI 0.41-2.25), a moderate (Tokyo grade II) more than doubled the risk (OR 2.41 95 % CI 1.21-4.80). Severe cholecystitis (Tokyo grade III) had a close to significant eightfold increase in risk (OR 8.43 95 % CI 0.97-72.9). The intention to use intraoperative cholangiography reduced injury risk by 52 % (OR 0.48, 95 % CI 0.29-0.81). CONCLUSIONS: Patients with on-going acute cholecystitis had twice the risk of sustaining a biliary lesion compared to patients without acute cholecystitis. There was a relation between the Tokyo guidelines severity grading of acute cholecystitis and injury risk and the intention to use intraoperative cholangiography halved the risk of reconstructed bile duct injury during cholecystectomy.

High p53 protein expression in therapy-related myeloid neoplasms is associated with adverse karyotype and poor outcome.

Identification of p53-positive cells by immunohistochemistry in bone marrow from primary myelodysplastic syndrome patients correlates with the presence of TP53 mutations and poor prognosis. Mutations in the tumor suppressor gene TP53 are more frequent in therapy-related acute myeloid leukemia and myelodysplastic syndrome than in de novo disease, but the role of p53 immunohistochemistry in the therapy-related setting has not been specifically investigated. We studied p53 protein immunoreactivity in bone marrow biopsies of therapy-related myeloid neoplasms and correlated protein expression with TP53 mutation status, clinicopathologic features and outcome. We first studied 32 patients with therapy-related acute myeloid leukemia and 63 patients with therapy-related myelodysplastic syndrome/chronic myelomonocytic leukemia from one institution and then validated our results in a separate group of 32 patients with therapy-related acute myeloid leukemia and 56 patients with therapy-related myelodysplastic syndrome from a different institution. Strong p53 immunostaining in ≥1% of bone marrow cells was highly predictive of a TP53 gene mutation (P<0.0001) and was strongly associated with a high-risk karyotype (P<0.0001). The presence of ≥1% p53 strongly positive cells was associated with poorer overall and disease-specific survival, particularly in the subset of patients treated with stem-cell transplantation. In a multivariable Cox regression model, the presence of ≥1% p53 strongly expressing cells was an independent prognostic marker for overall survival in both cohorts, with hazard ratios of 3.434 (CI: 1.751-6.735, P<0.0001) and 3.156 (CI: 1.502-6.628, P=0.002). Our data indicate that p53 protein expression, evaluated in bone marrow biopsies by a widely available immunohistochemical method, prognostically stratifies patients with therapy-related myeloid neoplasms independent of other risk factors. p53 immunostaining thus represents an easily applicable method to assess risk in therapy-related acute myeloid leukemia/myelodysplastic syndrome patients.

Variant Profiling of Candidate Genes in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Variant profiling is crucial for developing personalized treatment and elucidating the etiology of this disease. METHODS: Patients with PDAC undergoing surgery from 2007 to 2012 (n = 73) were followed from diagnosis until death or the end of the study. We applied an anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) method to a panel of 65 selected genes and assessed analytical performance by sequencing a quantitative multiplex DNA reference standard. In clinical PDAC samples, detection of low-level KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations was validated by allele-specific PCR and digital PCR. We compared overall survival of patients according to KRAS mutation status by log-rank test and applied logistic regression to evaluate the association between smoking and tumor variant types. RESULTS: The AMP-based NGS method could detect variants with allele frequencies as low as 1% given sufficient sequencing depth (>1500×). Low-frequency KRAS G12 mutations (allele frequency 1%-5%) were all confirmed by allele-specific PCR and digital PCR. The most prevalent genetic alterations were in KRAS (78% of patients), TP53 (tumor protein p53) (25%), and SMAD4 (SMAD family member 4) (8%). Overall survival in T3-stage PDAC patients differed among KRAS mutation subtypes (P = 0.019). Transversion variants were more common in ever-smokers than in never-smokers (odds ratio 5.7; 95% CI 1.2-27.8). CONCLUSIONS: The AMP-based NGS method is applicable for profiling tumor variants. Using this approach, we demonstrated that in PDAC patients, KRAS mutant subtype G12V is associated with poorer survival, and that transversion variants are more common among smokers.

Monitoring Hepatocellular Carcinoma Using Tumor Content in Circulating Cell-Free DNA.

PURPOSE: The objective of the study was to evaluate the use of tumor content in circulating cell-free DNA (ccfDNA) for monitoring hepatocellular carcinoma (HCC) throughout its natural history. EXPERIMENTAL DESIGN: We included 67 patients with hepatitis B virus-related HCC, of whom 17 had paired pre- and posttreatment samples, and 90 controls. Additionally, in a prospective cohort with hepatitis B virus surface antigen-positive participants recruited in 2012 and followed up biannually with blood sample collections until 2019, we included 270 repeated samples before diagnosis from 63 participants who later developed HCC (pre-HCC samples). Shallow whole-genome sequencing and the ichorCNA method were used to analyze genome-wide copy number and tumor content in ccfDNA. RESULTS: High tumor content was associated with advanced tumor stage (P < 0.001) and poor survival after HCC diagnosis [HR = 12.35; 95% confidence interval (CI) = 1.413-107.9; P = 0.023]. Tumor content turned negative after surgery (P = 0.027), whereas it remained positive after transarterial chemoembolization treatment (P = 0.578). In non-HCC samples, the mean tumor content (±SD) was 0.011 (±0.007) and had a specificity of 97.8% (95% CI = 92.2%-99.7%). In pre-HCC samples, the tumor content increased from 0.014 at 4 years before diagnosis to 0.026 at 1 year before diagnosis. The sensitivity of tumor content in detecting HCC increased from 22.7% (95% CI = 11.5%-37.8%) within 1 year before diagnosis to 30.4% (95% CI = 13.2%-52.9%) at the Barcelona Clinic Liver Cancer (BCLC) stage 0/A, 81.8% (95% CI = 59.7%-94.8%) at stage B, and 95.5% (95% CI = 77.2%-99.9%) at stage C. CONCLUSIONS: The tumor content in ccfDNA is correlated with tumor burden and may help in monitoring HCC 1 yearearlier than clinical diagnosis and in predicting patient prognosis.

Circulating DNA genome-wide fragmentation in early detection and disease monitoring of hepatocellular carcinoma.

Genome-wide circulating cell-free DNA (ccfDNA) fragmentation for cancer detection has been rarely evaluated using blood samples collected before cancer diagnosis. To evaluate ccfDNA fragmentation for detecting early hepatocellular carcinoma (HCC), we first modeled and tested using hospitalized HCC patients and then evaluated in a population-based study. A total of 427 samples were analyzed, including 270 samples collected prior to HCC diagnosis from a population-based study. Our model distinguished hospital HCC patients from controls excellently (area under curve 0.999). A high ccfDNA fragmentation score was highly associated with an advanced tumor stage and a shorter survival. In evaluation, the model showed increasing sensitivities in detecting HCC using 'pre-samples' collected ≥4 years (8.3%), 3-4 years (20.0%), 2-3 years (31.0%), 1-2 years (35.0%), and 0-1 year (36.4%) before diagnosis. These findings suggested ccfDNA fragmentation is sensitive in clinical HCC detection and might be helpful in screening early HCC.

Erector spinae plane block versus caudal block for postoperative analgesia in pediatric patients undergoing inguinal hernia repair: a randomized controlled trial.

BACKGROUND: Erector spinae plane block is a promising strategy for pain management in some settings. However, the effectiveness of erector spinae plane block versus caudal block in pediatric inguinal hernia repair has yet to be formally investigated. METHODS: One hundred and two patients aged 2-5 years undergoing unilateral open inguinal hernia repair randomly received unilateral erector spinae plane block (0.2% ropivacaine 0.5 mL kg-1), caudal block (0.2% ropivacaine 1 mL kg-1), or no block. The primary outcome was time to the first rescue analgesia, defined as the interval from the end of surgery to the Face, Legs, Activity, Cry, and Consolability scale greater than three. Secondary outcomes included the number of patients requiring rescue analgesia, the area under the curve of pain scores over time, satisfaction of guardians, and adverse events. RESULTS: The median time to the first rescue analgesia was longer in the erector spinae plane block group than in the caudal block group [10.0 h (interquartile range, 6.6-24.0 h) vs. 5.0 h (interquartile range, 2.9-7.3 h); p < .001]. The Cox regression model demonstrated that the risk of postoperative rescue analgesia requirement was 0.38 in children receiving erector spinae plane block compared with caudal block (95% confidence interval 0.23-0.64; p < .001). Additionally, the area under the curve of the pain scores over time was lower in the erector spinae plane block group than in the caudal block group (44.3 [36.6-50.7] vs. 59.0 [47.1-64.5]; p < .001). CONCLUSIONS: Erector spinae plane block provided superior postoperative analgesia compared to caudal block in children undergoing inguinal hernia repair.Trial registration: Chinese Clinical Trial Registry; ChiCTR2100048303.

Erector spinae plane block (ESPB) is beneficial for postoperative analgesia in children undergoing inguinal hernia repair.Ultrasound-guided ESPB provided superior analgesia efficacy to caudal block in the pediatric population.ESPB is an attractive strategy for pain management after lower abdominal surgical procedures.

Sex Differences in Long-term Outcome of Prenatal Exposure to Excess Glucocorticoids-Implications for Development of Psychiatric Disorders.

Exposure to prenatal insults, such as excess glucocorticoids (GC), may lead to pathological outcomes, including neuropsychiatric disorders. The aim of the present study was to investigate the long-term effects of in utero exposure to the synthetic GC analog dexamethasone (Dex) in adult female offspring. We monitored spontaneous activity in the home cage under a constant 12 h/12 h light/dark cycle, as well as the changes following a 6-h advance of dark onset (phase shift). For comparison, we re-analysed data previously recorded in males. Dex-exposed females were spontaneously more active, and the activity onset re-entrained slower than in controls. In contrast, Dex-exposed males were less active, and the activity onset re-entrained faster than in controls. Following the phase shift, control females displayed a transient reorganisation of behaviour in light and virtually no change in dark, while Dex-exposed females showed limited variations from baseline in both light and dark, suggesting weaker photic entrainment. Next, we ran bulk RNA-sequencing in the suprachiasmatic nucleus (SCN) of Dex and control females. SPIA pathway analysis of ~ 2300 differentially expressed genes identified significantly downregulated dopamine signalling, and upregulated glutamate and GABA signalling. We selected a set of candidate genes matching the behaviour alterations and found consistent differential regulation for ~ 73% of tested genes in SCN and hippocampus tissue samples. Taken together, our data highlight sex differences in the outcome of prenatal exposure to excess GC in adult mice: in contrast to depression-like behaviour in males, the phenotype in females, defined by behaviour and differential gene expression, is consistent with ADHD models.

Single-cell RNA sequencing reveals maturation trajectory in human pluripotent stem cell-derived cardiomyocytes in engineered tissues.

Cardiac in vitro models have become increasingly obtainable and affordable with the optimization of human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) differentiation. However, these CMs are immature compared to their in vivo counterparts. Here we study the cellular phenotype of hPSC-CMs by comparing their single-cell gene expression and functional profiles in three engineered cardiac tissue configurations: human ventricular (hv) cardiac anisotropic sheet, cardiac tissue strip, and cardiac organoid chamber (hvCOC), with spontaneously aggregated 3D cardiac spheroids (CS) as control. The CM maturity was found to increase with increasing levels of complexity of the engineered tissues from CS to hvCOC. The contractile components are the first function to mature, followed by electrophysiology and oxidative metabolism. Notably, the 2D tissue constructs show a higher cellular organization whereas metabolic maturity preferentially increases in the 3D constructs. We conclude that the tissue engineering models resembling configurations of native tissues may be reliable for drug screening or disease modeling.