Hybridization Sequencing Research Articles

Emergence of a novel hybrid mcr-1-bearing plasmid in an NDM-7-producing ST167 Escherichia coli strain of clinical origin.

Colistin is considered as an antibiotic of ‘last resort’ for the treatment of lethal infections caused by carbapenem-resistant Enterobacterales (CRE), dissemination of plasmid-borne colistin resistance gene mcr-1, particularly into CRE, resulting in the emergence of strains that approach pan-resistance. A wide variety of plasmid types have been reported for carrying mcr-1. Among which, large IncHI2-type plasmids were multidrug-resistant (MDR) plasmids harbored multiple resistance determinants in addition to mcr-1. Herein, we characterized a novel hybrid IncHI2-like mcr-1-bearing plasmid in an NDM-7-producing ST167 Escherichia coli strain EC15-50 of clinical origin. Antimicrobial susceptibility testing showed E. coli EC15-50 exhibited an extensively drug-resistant (XDR) profile that only susceptible to amikacin and tigecycline. S1-PFGE, Southern hybridization and Whole-genome Sequencing (WGS) analysis identified a 46,161 bp blaNDM-7-harboring IncX3 plasmid pEC50-NDM7 and a 350,179 bp mcr-1-bearing IncHI2/HI2A/N/FII/FIA plasmid pEC15-MCR-50 in E. coli EC15-50. Sequence detail analysis revealed the type IV coupling protein (T4CP) gene was absent on pEC15-MCR-50, explaining that pEC15-MCR-50 was a non-conjugative plasmid. Comparative genetic analysis indicated the hybrid plasmid pEC15-MCR-50 was probably originated from pXGE1mcr-like IncHI2/HI2A/N plasmid and pSJ_94-like IncFII/FIA plasmid, and generated as a result of a replicative transposition process mediated by IS26. Currently, the prevalent mcr-1-carrying IncHI2 plasmids were rarely reported to be fused with other plasmids. The identification of the novel hybrid plasmid pEC15-MCR-50 in this study highlighted the importance of close surveillance for the emergence and dissemination of such fusion MDR plasmids, particularly in NDM-producing Enterobacterales.

Deletion of Tfam in Prx1-Cre expressing limb mesenchyme results in spontaneous bone fractures.

Osteoblasts require substantial amounts of energy to synthesize the bone matrix and coordinate skeleton mineralization. This study analyzed the effects of mitochondrial dysfunction on bone formation, nano-organization of collagen and apatite, and the resultant mechanical function in mouse limbs. Limb mesenchyme-specific Tfam knockout (Tfamf/f;Prx1-Cre: Tfam-cKO) mice were analyzed morphologically and histologically, and gene expressions in the limb bones were assessed by in situ hybridization, qPCR, and RNA sequencing (RNA-seq). Moreover, we analyzed the mitochondrial function of osteoblasts in Tfam-cKO mice using mitochondrial membrane potential assay and transmission electron microscopy (TEM). We investigated the pathogenesis of spontaneous bone fractures using immunohistochemical analysis, TEM, birefringence analyzer, microbeam X-ray diffractometer and nanoindentation. Forelimbs in Tfam-cKO mice were significantly shortened from birth, and spontaneous fractures occurred after birth, resulting in severe limb deformities. Histological and RNA-seq analyses showed that bone hypoplasia with a decrease in matrix mineralization was apparent, and the expression of type I collagen and osteocalcin was decreased in osteoblasts of Tfam-cKO mice, although Runx2 expression was unchanged. Decreased type I collagen deposition and mineralization in the matrix of limb bones in Tfam-cKO mice were associated with marked mitochondrial dysfunction. Tfam-cKO mice bone showed a significantly lower Young's modulus and hardness due to poor apatite orientation which is resulted from decreased osteocalcin expression. Mice with limb mesenchyme-specific Tfam deletions exhibited spontaneous limb bone fractures, resulting in severe limb deformities. Bone fragility was caused by poor apatite orientation owing to impaired osteoblast differentiation and maturation.

PGT for structural chromosomal rearrangements in 300 couples reveals specific risk factors but an interchromosomal effect is unlikely

Research questionWhat factors affect the proportion of chromosomally balanced embryos in structural rearrangement carriers? Is there any evidence for an interchromosomal effect (ICE)? DesignPreimplantation genetic testing outcomes of 300 couples (198 reciprocal, 60 Robertsonian, 31 inversion and 11 complex structural rearrangement carriers) were assessed retrospectively. Blastocysts were analysed either by array-comparative genomic hybridization or next-generation sequencing techniques. ICE was investigated using a matched control group and sophisticated statistical measurement of effect size (φ). Results300 couples underwent 443 cycles; 1835 embryos were analysed and 23.8% were diagnosed as both normal/balanced and euploid. The overall cumulative clinical pregnancy and live birth rates were 69.5% and 55.8%, respectively. Complex translocations and female age (≥35) were found to be risk factors associated with lower chance of having a transferable embryo (P < 0.001). Based on analysis of 5237 embryos, the cumulative de-novo aneuploidy rate was lower in carriers compared to controls (45.6% versus 53.4%, P < 0.001) but this was a ‘negligible’ association (φ < 0.1). A further assessment of 117,033 chromosomal pairs revealed a higher individual chromosome error rate in embryos of carriers compared to controls (5.3% versus 4.9%), which was also a ‘negligible’ association (φ < 0.1), despite a P-value of 0.007. ConclusionsThese findings suggest that rearrangement type, female age and sex of the carrier have significant impacts on the proportion of transferable embryos. Careful examination of structural rearrangement carriers and controls indicated little or no evidence for an ICE. This study helps to provide a statistical model for investigating ICE and an improved personalized reproductive genetics assessment for structural rearrangement carriers.

A novel free-breathing abdominal RAVE T2/T1 hybrid MRI sequence in patients with cystic fibrosis: Preliminary results

ObjectivesPatients with cystic fibrosis (CF) increasingly require imaging for the diagnosis of abdominal complications. We prospectively evaluated the image quality and signal-to-noise ratio (SNR) of a modern radial volumetric encoding (RAVE) T2/T1 hybrid sequence for abdominal magnetic resonance imaging (MRI). RAVET2/T1 is a three-dimensional radial sequence with fat saturation and blood flow suppression that acquires T2- and T1-weighted contrasts in one scan in an identical slice position during free-breathing. MethodsSixteen CF patients underwent axial T2 HASTE (1000 ms/93 ms TR/TE), T1 DIXON (6.8 ms/2.4 ms/4.8 ms TR/TE1/TE2), and RAVE T2/T1 hybrid sequence (1200 ms/1.7 ms/3.3 ms/4.9 ms/102 ms TR/TE1/TE2/TE3/TE4) of the upper abdomen at 1.5 Tesla. The SNR values in six different regions were assessed and compared using the Wilcoxon signed-rank test. The image quality criteria were rated on a 5-point Likert scale. ResultsIn all regions, the SNR was significantly higher in the T2 weighted aspect of the RAVE T2/T1 hybrid sequence compared to T2 HASTE (p < 0.05) and significantly lower in the T1 weighted in-phase aspect of the RAVE T2/T1 hybrid sequence compared to the T1 DIXON sequence (p < 0.05).Qualitatively the T2 weighted aspect of the RAVE T2/T1 hybrid sequence was rated significantly higher than the T2 HASTE in 6 of 7 categories (p < 0.05) and the T1 weighted in-phase aspect of the RAVE T2/T1 hybrid sequence was rated significantly higher than the T1 DIXON in 2 of 6 categories (p < 0.05). ConclusionsThe abdominal radial RAVE T2/T1 hybrid sequence provided higher image quality and SNR than the T2HASTEsequence. Together with increased robustness against motion artifacts, the RAVE T2/T1 hybrid sequence appears to be a good tool for abdominal imaging in CF patients.

Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single-cell resolution

The Mexican axolotl (Ambystoma mexicanum) is a well-established tetrapod model for regeneration and developmental studies. Remarkably, neotenic axolotls may undergo metamorphosis, a process that triggers many dramatic changes in diverse organs, accompanied by gradually decline of their regeneration capacity and lifespan. However, the molecular regulation and cellular changes in neotenic and metamorphosed axolotls are still poorly investigated. Here, we develop a single-cell sequencing method based on combinatorial hybridization to generate a tissue-based transcriptomic landscape of the neotenic and metamorphosed axolotls. We perform gene expression profiling of over 1 million single cells across 19 tissues to construct the first adult axolotl cell landscape. Comparison of single-cell transcriptomes between the tissues of neotenic and metamorphosed axolotls reveal the heterogeneity of non-immune parenchymal cells in different tissues and established their regulatory network. Furthermore, we describe dynamic gene expression patterns during limb development in neotenic axolotls. This system-level single-cell analysis of molecular characteristics in neotenic and metamorphosed axolotls, serves as a resource to explore the molecular identity of the axolotl and facilitates better understanding of metamorphosis.

Natural history of KBG syndrome in a large European cohort.

KBG syndrome (KBGS) is characterized by distinctive facial gestalt, short stature and variable clinical findings. With ageing, some features become more recognizable, allowing a differential diagnosis. We aimed to better characterize natural history of KBGS. In the context of a European collaborative study, we collected the largest cohort of KBGS patients (49). A combined array- based Comparative Genomic Hybridization and next generation sequencing (NGS) approach investigated both genomic Copy Number Variants and SNVs. Intellectual disability (ID) (82%) ranged from mild to moderate with severe ID identified in two patients. Epilepsy was present in 26.5%. Short stature was consistent over time, while occipitofrontal circumference (median value: -0.88 SD at birth) normalized over years. Cerebral anomalies, were identified in 56% of patients and thus represented the second most relevant clinical feature reinforcing clinical suspicion in the paediatric age when short stature and vertebral/dental anomalies are vague. Macrodontia, oligodontia and dental agenesis (53%) were almost as frequent as skeletal anomalies, such as brachydactyly, short fifth finger, fifth finger clinodactyly, pectus excavatum/carinatum, delayed bone age. In 28.5% of individuals, prenatal ultrasound anomalies were reported. Except for three splicing variants, leading to a premature termination, variants were almost all frameshift. Our results, broadening the spectrum of KBGS phenotype progression, provide useful tools to facilitate differential diagnosis and improve clinical management. We suggest to consider a wider range of dental anomalies before excluding diagnosis and to perform a careful odontoiatric/ear-nose-throat (ENT) evaluation in order to look for even submucosal palate cleft given the high percentage of palate abnormalities. NGS approaches, following evidence of antenatal ultrasound anomalies, should include ANKRD11.

Influence of hybridization on the mechanical and dynamic mechanical properties of aramid/S2-glass hybrid laminates

This work investigates the effect of hybridization and stacking sequence on the response of S2-glass/aramid/epoxy interply hybrid laminates, focusing on flexural, short beam, dynamic-mechanical results and, especially, quasi-static indentation (QSI) properties. Different laminates of comparable thickness were manufactured by vacuum infusion, one with only aramid fabrics ([K]4S), one with only S2-glass fabrics ([G]8S) and six interply hybrids, namely [G6K5], i.e. six S2-glass layers on top of five aramid layers (asymmetric), [G8K4] [G10K3], [K2G4]S, [G2K]S2 and [G4K2]S. QSI tests were used to determine absorbed energy, maximum energy and peak load, and the in-plane damage was analyzed. Significant differences in mechanical and dynamic-mechanical properties (pairing effect) were observed for the [K2G4]S, [G2K]2S, [G4K2]S and [G8K4] hybrids, and the adhesion factor (A) presented the closest correlation with energy absorption capacity of the laminates. The results have shown that hybridization of aramid composites with S2-glass may enhance impact absorption, and a positive hybrid effect was observed for the laminates with S2-glass fibers on the top surface, [G6K5], with peak load and absorbed energy (9.9 kN and 55 J, respectively) higher than that of the pure laminates [G]8S (5.9 kN and 41 J) and [K]4S (8.6 kN and 46 J).

Copy Number Variation Analysis of 5p Deletion Provides Accurate Prenatal Diagnosis and Reveals Candidate Pathogenic Genes

Objective5p deletion syndrome, that characterized by cat-like cry and peculiar timbre of voice, is believed to be one of the most common pathogenic copy number variations (CNVs). Variable critical regions on 5p involving a variety of genes contribute to the phenotypic heterogeneity without specific correlation. The objective of this study was to examine the genotype–phenotype correlation of 5p deletion syndrome, and to redefine 5p deletion syndrome relevant regions. In addition, we demonstrate the potential use of whole genome sequencing (WGS) to identify chromosomal breakpoints in prenatal diagnosis.MethodsThree families with women undergoing prenatal diagnosis and two children were recruited. Karyotyping, CNV-seq, fluorescence in situ hybridization, WGS, and Sanger sequencing were performed to identify the chromosomal disorder.ResultsWe reported three families and two children with CNVs of 5p deletion or combined 6p duplication. Five different sizes of 5p deletion were detected and their pathogenicity was determined, including 5p15.33-p15.31 [1–7,700,000, family1-variant of uncertain significance (VUS)], 5p15.33 (1–3,220,000, family 2-VUS), 5p15.33-p15.31 (1–7,040,000, family 3-VUS), 5p15.33-p15.31 (1–8,740,000, child 1-pathogenic) and 5p15.31-p15.1 (8,520,001–18,080,000, child 2-pathogenic). One duplication at 6p25.3-p24.3 (1–10,420,000) was detected and determined as likely pathogenic. The chromosomal breakpoints in family 3 were successfully identified by WGS.ConclusionSome critical genes that were supposed to be causative of the symptoms were identified. Relevant region in 5p deletion syndrome was redefined, and the chr5:7,700,000–8,740,000 region was supposed to be responsible for the cat-like cry. The great potential of WGS in detecting chromosomal translocations was demonstrated. Our findings may pave the way for further research on the prevention, diagnosis, and treatment of related diseases.

Clinical and genetic analysis of three children with 22q13 deletion syndrome

To explore the clinical and genetic characteristics of three children with 22q13 deletion syndrome. Clinical data were collected and copy number variations in the patients and their parents were detected by using array-based comparative genomic hybridization (aCGH) and copy number variation sequencing (CNV-seq). The DECIPHER, ClinGen, OMIM, PubMed and Gene Review databases were retrieved for pathogenicity analysis. The common phenotypes of the three children have included variable global developmental delay, among which speech delay was the most obvious. Patient 1 had abnormalities of corpus callosum shown by magnetic resonance imaging. Patient 2 had dental crowding, pale skin, thick palms, hypotonia, and other facial features. Patient 3 had the mildest symptoms including language dysfunction, which has caught up with the development and improved significantly. All of the three children had harbored de novo deletions of 22q13.33q13.33 region, which spanned 0.84 Mb, 8.70 Mb and 0.90 Mb and involved 37, 126, and 34 genes, respectively. Above finding has enriched the clinical and genetic characteristics of 22q13 deletion syndrome and laid a foundation for genetic counseling and prenatal diagnosis.

Corynebacterium matruchotii: A Confirmed Calcifying Bacterium With a Potentially Important Role in the Supragingival Plaque.

Corynebacterium matruchotii is a reported calcifying bacterium that can usually be isolated from dental calculus and induce mineralization in vitro. In recent years, based on in situ hybridization probe and sequencing technology, researchers have discovered the central “pillar” role of C. matruchotii in supragingival plaque, and many studies focused on bacterial interactions in the biofilm structure dominated by C. matruchotii have been conducted. Besides, C. matruchotii seems to be an indicator of “caries-free” oral status according to imaging and sequencing studies. Therefore, in this review, we summarize C. matruchotii ‘s role in supragingival plaque based on the structure, interactions, and potential connections with oral diseases.

Lymphoproliferative disorder involving body fluid: diagnostic approaches and roles of ancillary studies.

Lymphocyte-rich effusions represent benign reactive process or neoplastic condition. Involvement of lymphoproliferative disease in body cavity is not uncommon, and it often causes diagnostic challenge. In this review, we suggest a practical diagnostic approach toward lymphocyte-rich effusions, share representative cases, and discuss the utility of ancillary tests. Cytomorphologic features favoring neoplastic condition include high cellularity, cellular atypia/pleomorphism, monomorphic cell population, and frequent apoptosis, whereas lack of atypia, polymorphic cell population, and predominance of small T cells usually represent benign reactive process. Involvement of non-hematolymphoid malignant cells in body fluid should be ruled out first, followed by categorization of the samples into either small/medium-sized cell dominant or large-sized cell dominant fluid. Small/medium-sized cell dominant effusions require ancillary tests when either cellular atypia or history/clinical suspicion of lymphoproliferative disease is present. Large-sized cell dominant effusions usually suggest neoplastic condition, however, in the settings of initial presentation or low overall cellularity, ancillary studies are helpful for more clarification. Ancillary tests including immunocytochemistry, in situ hybridization, clonality test, and next-generation sequencing can be performed using cytologic preparations. Throughout the diagnostic process, proper review of clinical history, cytomorphologic examination, and application of adequate ancillary tests are key elements for successful diagnosis.

Metaphase II (MII) human oocytes with smooth endoplasmic reticulum clusters do not affect blastocyst euploid rate

ObjectiveTo investigate whether the rate of euploidy and pregnancy outcomes are affected by smooth endoplasmic reticulum clusters (SERc) and other metaphase II human oocyte dysmorphisms. Materials and methodsRetrospective analysis of the morphologies of metaphase II (MII) human oocytes, which had developed into 590 biopsied blastocysts derived from 109 patients that received preimplantation genetic testing for aneuploidies (PGT-A) cycles between March 2013 and December 2017. The euploid rate of blastocysts that originated from morphologically abnormal or normal oocytes were analyzed. The chromosome status of the blastocysts was determined and analyzed by array comparative genomic hybridization (aCGH) or next generation sequencing (NGS) following trophectoderm biopsy. ResultsAccording to the odds ratios obtained for each oocyte morphotype, no statistically significant relationship was found between oocyte dysmorphisms and euploid rate. Specifically, although SERc-positive oocytes had a higher rate of arrest at two pronuclei, or 2 PN (26.7% vs. 19.4%, p > 0.05), the blastocyst formation rate was not affected as compared with SERc-negative oocytes (40.0% vs. 38.6%, p > 0.05). Among nine euploid embryos derived from oocytes with SERc, three single euploid embryo transfers were performed, of which one resulted in blighted ovum, and two resulted in the births of two healthy, singleton term babies. ConclusionThe results presented here suggest that oocyte dysmorphisms do not affect the euploidy rate of the blastocyst. The occurrence of SERc in the oocyte does not seem to impair the developing blastocyst nor does it interfere with good embryo formation rate and euploid rate. Thus, the embryos derived from SERc-positive oocytes could still be considered for embryo transfer if there are no other embryos available.

P-357 Predicting live birth following single euploid frozen blastocyst transfer - does the journey matter?

Abstract Study question Does the number of available euploid embryos affect the success rate of the first single euploid frozen thawed embryo transfer (FTET)? Summary answer The live birth rate (LBR) following the first single euploid FTET is independent of the number of available euploid embryos. What is known already Preimplantation testing for aneuploidy (PGT-A) is a controversial laboratory technique as an embryo selection tool to increase LBR per transfer and reduce time to pregnancy. There have been many efforts to identify factors predictive of LBR following a euploid embryo transfer. The number of euploid embryos available is known to increase the expected cumulative LBR. However, there is lack of evidence whether the number of available euploid embryos is also significantly associated with chances of live birth for the first euploid FTET cycle. This is important for individualized patient counselling. Study design, size, duration A retrospective analysis of the first, single, euploid FTET of 506 women, between January 2015 to March 2020 following PGT-A was carried out. The indication for PGT-A was advanced maternal age, recurrent miscarriage or implantation failure. Embryos were biopsied on day 5 or 6 and tested with array comparative genetic hybridization or next generation sequencing. The FTET outcomes were compared based on the number of euploid embryos available. Participants/materials, setting, methods 198 women had one euploid embryo available (group 1), 120 women had two (group 2) and 188 women had three or more (group 3). Baseline characteristics were analysed for all participants to adjust for confounders. Reproductive outcomes such as pregnancy rate (PR), clinical miscarriage rate and LBR were compared between the 3 groups for the first euploid FTET. Univariate analysis with Kruskal-Wallis H, chi-square and multivariate logistic regression was performed with SPSS. Main results and the role of chance Baseline characteristics were similar between the groups including age at egg collection (EC), body mass index, use of partner or donor sperm, method of insemination, number of ECs, endometrial preparation protocol and endometrial thickness prior to transfer. Women with three or more euploid embryos were younger at the time of EC compared to groups 1 and 2 (mean age 37 vs 39 and 38). The number of oocytes per EC (15 vs 11 and 11, p &amp;lt; 0.001), the total number of oocytes following batching cycles (20 vs 15 and 16, p &amp;lt; 0.001) and the number of blastocysts biopsied (8 vs 4 and 5, p &amp;lt; 0.001) were significantly higher for Group 3 vs Group 1 and Group 2, respectively. Group 1 embryos were less frequently of excellent and very good morphology and less frequently biopsied on day 5 vs 6. Univariate analysis suggests Group 3 had statistically higher PR and LBR compared to Group 1, but similar miscarriage rate. However, when applying a multivariate logistic regression model, the LBR after first euploid FTET was independent of the number of euploid embryos available but was influenced by age at EC (p = 0.006) and biopsy day (Day 6 vs day 5, OR 0.53, 95% CI 0.33-0.85, p = 0.008). Limitations, reasons for caution The main limitation of this study is its retrospective nature performed in a single centre, and therefore vulnerable to bias and confounding factors that may have not been accounted for. Wider implications of the findings Our findings suggest that the success rate of the first euploid FTET does not depend on the number of euploid embryos but is influenced by the age at EC and the embryo stage at biopsy. This could be useful when counselling women with a low number of euploid embryos available. Trial registration number Not Applicable

Spent Fuel Pool Dose Rate Calculations Using Point Kernel and Hybrid Deterministic-Stochastic Shielding Methods

This paper presents comparison of the Krško Power Plant simplified Spent Fuel Pool (SFP) dose rates using different computational shielding methodologies. The analysis was performed to estimate limiting gamma dose rates on wall mounted level instrumentation in case of significant loss of cooling water. The SFP was represented with simple homogenized cylinders (point kernel and Monte Carlo (MC)) or cuboids (MC) using uranium, iron, water, and dry-air as a bulk region materials. The pool is divided on the old and new section where the old one has three additional subsections representing fuel assemblies (FAs) with different burnup/cooling time (60 days, 1 year and 5 years). The new section represents the FAs with the cooling time of 10 years. The time dependent fuel assembly isotopic composition was calculated using ORIGEN2 code applied to the depletion of one of the fuel assemblies present in the pool (AC-29). The source used in Microshield calculation is based on imported isotopic activities. The time dependent photon spectrum with total source intensity from Microshield multigroup point kernel calculations was then prepared for two hybrid deterministic-stochastic sequences. One is based on SCALE6.2b3/MAVRIC (Monaco and Denovo) methodology and another uses Monte Carlo code MCNP6.1.1b and ADVANTG3.0.1. code. Even though this model is a fairly simple one, the layers of shielding materials are thick enough to pose a significant shielding problem for MC method without the use of effective variance reduction (VR) technique. For that purpose the ADVANTG code was used to generate VR parameters for the MCNP fixed-source calculation using continuous energy transport. ADVATNG employs a deterministic forward-adjoint transport solver Denovo which implements CADIS/FWCADIS methodology. Denovo uses a structured, Cartesian-grid SN solver based on the Koch- Baker-Alcouffe parallel transport sweep algorithm across x-y domain blocks. This was our first application of ANDVANTG/MCNP hybrid sequence for this type of calculation and the results where compared to SCALE/MAVRIC sequence which we regularly use for similar calculations. The comparison of gamma dose rates on different point detector locations (central above pool and at the top of pool periphery) showed a good agreement between Microshield (point-kernel) and deterministic-stochastic shielding methodologies for the cylindrical approximation of the pool geometry. More complicated cases for model with multi-source option and for cuboids showed very good agreement between SCALE/MAVRIC and ANDVANTG/MCNP calculations.

Genomic Insights of Enterococcus faecium UC7251, a Multi-Drug Resistant Strain From Ready-to-Eat Food, Highlight the Risk of Antimicrobial Resistance in the Food Chain.

The presence of multi-drug resistant (MDR) bacteria in ready-to-eat foods comprises a threat for public health due to their ability to acquire and transfer antibiotic-resistant determinants that could settle in the microbiome of the human digestive tract. In this study, Enterococcus faecium UC7251 isolated from a fermented dry sausage was characterized phenotypically and genotypically to hold resistance to multiple antibiotics including aminoglycosides, macrolides, β-lactams, and tetracyclines. We further investigated this strain following a hybrid sequencing and assembly approach (short and long reads) and determined the presence of various mobile genetic elements (MGEs) responsible of horizontal gene transfer (HGT). On the chromosome of UC7251, we found one integrative and conjugative element (ICE) and a conjugative transposon Tn916-carrying tetracycline resistance. UC7251 carries two plasmids: one small plasmid harboring a rolling circle replication and one MDR megaplasmid. The latter was identified as mobilizable and containing a putative integrative and conjugative element-like region, prophage sequences, insertion sequences, heavy-metal resistance genes, and several antimicrobial resistance (AMR) genes, confirming the phenotypic resistance characteristics. The transmissibility potential of AMR markers was observed through mating experiments, where Tn916-carried tetracycline resistance was transferred at intra- and inter-species levels. This work highlights the significance of constant monitoring of products of animal origin, especially RTE foodstuffs, to stimulate the development of novel strategies in the race for constraining the spread of antibiotic resistance.

RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.

Secretory Cells Are the Primary Source of pIgR in Small Airways.

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice used immunostaining, primary murine tracheal epithelial cell culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from nontypeable Haemophilus influenzae was evaluated invitro. We found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and nontypeable H. influenzae-secreted proteases are both capable of degrading SIgA invitro and may also contribute to a deficient SIgA immunobarrier in COPD. Loss of the SIgA immunobarrier in small airways of patients with severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.

Experimental study on the low-velocity impact response of inter-ply S2-glass/aramid woven fabric hybrid laminates

This paper presents an experimental evaluation of the effect of hybridization and stacking sequence on the low-velocity impact response of aramid/S2-glass/epoxy hybrid laminates. Plain-weave Kevlar ®29 and satin S2-glass fabrics were used and the laminates were manufactured by vacuum infusion moulding. Eight laminate configurations were studied, including two single-fibre samples (pure aramid or pure S2-glass) and six interply hybrids. Low-velocity impact tests were performed with three different impact energies (19 J, 37 J, 72 J) and significant variations in impact behaviour were observed among the laminates. Response Surface Methodology (RSM) was employed to investigate the effect of hybridization on impact performance, focusing on greater absorbed energy and smaller back face deformation. Hybridization led to significant changes in failure morphology and the combination of aramid and S2-glass laminates enhanced the impact performance, especially for up to 49.5 J impact energy, while the pure S2-glass laminate performed better for higher impact energies.

PATH-01. Infant-Type Hemispheric Glioma: New Molecular Alterations and Precision-Medicine Treatment

BACKGROUND: Infant-type hemispheric glioma, harboring alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET, is a new subtype of Pediatric-type diffuse high-grade gliomas in the 2021 WHO classification of CNS tumors. It has important clinical therapeutic value with specialized therapeutic drugs. Here, we presented 3 cases of infant-type hemispheric glioma. Patient1 with EML4-ALK fusion which often appeared in lung cancer, the other 2 patients have new molecular alterations which has not been reported before (Patient2 has both NTRK1-TP53/TP53-NTRK1 fusions and p53 protein showed characteristic cytoplasm positive; Patient3 presented a brand-new ALK-QKI fusion combined with ALK mutation and focal SMARCB1 deletion. All these 3 cases received corresponding targeted therapy and have a good recovery and normal neurologic function till now.METHOD: Immunohistochemistry, fluorescent in situ hybridization and whole-transcriptome sequencing. RESULTS: Case 18 months, male, right semiovale center occupation. Histopathology: High-grade neuroepithelial neoplasm. IHC: GFAP(only few cells+), Olig2(-), S100(+), CD56(+), Syn(-), NSE(focal+), NeuN(-), CD34(-), INI-1(+), Ki67(10%+). Characteristic Molecular Information: EML4-ALK fusion. Follow-up: 15 months, alive. Case 2: 3 years, female, insular lobe occupation.Histopathology: Gliosarcoma. IHC: GFAP(partly+), Olig2(partly+), Vimentin(+), P53(cytoplasm+), pan-TRK(+), Ki67(25%+). Reticular fiber staining showed biphasic tissue pattern with reticulin-rich sarcomatous and reticulin-free gliomatous elements. Characteristic Molecular Information: NTRK1-TP53 and TP53-NRTK1 fusion. Follow-up: 27 months, alive. Case 3: 3 years, male, left parietal occipital lobe occupation. Histopathology: GBM and AT/RT. IHC: GFAP(partly+), Olig2(partly+), INI-1(partly-), BRG1(+), SYN(-), CD34(-), BRAF(-), S100(-), CK(-), H3K27M(-), IDH1(-), P53(40%+), ATRX(+), pan-TRK(-), ALK(+), Ki67(30%+). Characteristic Molecular Information: ALK mutation, ALK-QKI fusion, RAD51C mutation and focal SMARCB1(INI-1) deletion. Follow-up: 14 months, alive. CONCLUSION: Infant-type hemispheric glioma is a special kind of glioma, which is particularly suitable for precision-medicine treatment approaches. Their overall survival is good compared with other three pHGG subtype.

HER2 in uterine carcinosarcoma: Testing platforms and implications for targeted therapy.

5590 Background: Uterine carcinosarcomas (UCS) are rare, aggressive tumors accounting for 5% of all uterine cancers. Recurrence rates are high and 5-year survival is only 18-39%. HER2 is an emerging prognostic and therapeutic target in uterine cancer including UCS. Testing algorithms and platforms in breast and gastric cancers are well studied and validated, but optimal HER2 testing in uterine cancer is not yet established. We aimed to determine HER2 prevalence in UCS and the concordance of chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), and next generation sequencing (NGS) platforms to aid in the development of UCS specific testing guidelines. We also evaluated the rate of downstream mutations and immune biomarkers that may affect response to HER2 directed therapy. Methods: Eight hundred and seventy-five UCS tumor samples (primary 81.4%; metastatic 17.1%; unknown 1.5%) were analyzed using NGS (592, NextSeq; WES, NovaSeq, Caris Life Sciences, Phoenix, AZ). A subset of tumors with HER2 positivity were tested with IHC (4B5, Ventana) and/or CISH (INFORM DUAL HER2 ISH Assay, Ventana) based on 2018 ASCO/CAP HER2 Breast Cancer guidelines. Amplification of ERBB2/HER2 by NGS used a copy number cut-off &gt;6. PD-L1 expression was analyzed by IHC (SP142, positive cut-off ≥1%, Ventana). Tumor mutational burden (TMB) was measured by counting all somatic mutations found per tumor (TMB high cut-off &gt; 10 mutations per Mb). Microsatellite instability (MSI) was tested by fragment analysis (FA), IHC and NGS. Results: Rates of HER2 positivity were 3.4% (28/820) by NGS. Among the IHC/CISH-tested cohort, 7.2% was positive (10/139) by IHC, and 19.5% (27/133) by CISH. In the 105 samples tested with both IHC and CISH, the concordance was 100%. Specifically, 9/105 patients (8.6%) were IHC+/CISH+ and 96 patients (91.4%) were IHC-/CISH-. The concordance between CISH and NGS (N = 127) was 90.6% (sensitivity 100% and specificity 89.3%). Common gene alterations in CISH HER2+ UCS tumors that may implicate resistance to HER2 targeted therapy included mutations in TP53 (100%), FBXW7 (22.2%), PIK3CA (33.3%), PIK3R1 (18.5%), PTEN (3.7%) and KRAS (3.7%) and gene amplification of KRAS (11.1%). CISH+ HER2+ UCS tumors had low immunotherapy biomarker prevalence (0% MSI-H, 0% TMB high, 7.7% PD-L1+). Conclusions: Increased HER2 positivity was detected via CISH testing compared to IHC and NGS, which may reflect the heterogeneity of HER2 amplification due to mixed histology between the sarcoma and carcinoma portion of the tumor. High concordance rates were observed between CISH and IHC. These testing platforms need to be validated by response to HER2 targeted therapies in order to develop UCS specific testing guidelines.