Acetylation regulates the nucleocytoplasmic distribution and oncogenic function of karyopherin alpha 2 in lung adenocarcinoma.

Karyopherin subunit alpha 2 (KPNA2, importin α1) is a nucleoplasmic protein responsible for the nuclear import of proteins with classical nuclear localization signals. Aberrant nuclear accumulation of KPNA2 has been observed in numerous cancer tissues. AMP-activated protein kinase (AMPK) is involved in the phosphorylation and acetylation of KPNA2 in enterocytes. However, the impact of these post-translational modifications on modulating the nucleocytoplasmic distribution of KPNA2 and its oncogenic role remain unclear. Unlike nuclear accumulation of wild-type KPNA2, which promoted lung cancer cell migration, KPNA2 Lys22 acetylation-mimicking mutations (K22Q and K22Q/S105A) prevented nuclear localization of KPNA2 and reduced the cell migration ability. Cytosolic KPNA2 K22Q interacted with and restricted the nuclear entry of E2F transcription factor 1 (E2F1), an oncogenic cargo protein of KPNA2, in lung cancer cells. Intriguingly, the AMPK activator EX229 promoted the nuclear export of KPNA2 S105A. However, the CBP/p300 inhibitor CCS-1477 abolished this phenomenon, suggesting that CBP/p300-mediated acetylation of KPNA2 promoted KPNA2 nuclear export in lung cancer cells. Collectively, our findings suggest that the CBP/p300 positively regulates KPNA2 acetylation, which enhances its cytosolic localization and suppresses its oncogenic activity in lung cancer.

Global Genome Diversity and Recombination in Mycoplasma pneumoniae.

Genomic changes in Mycoplasma pneumoniae caused by adaptation to environmental or ecologic pressures are poorly understood. We collected M. pneumoniae from children who had confirmed pneumonia in Taiwan during 2017-2020. We used whole-genome sequencing to compare these isolates with a worldwide collection of current and historical clinical strains for characterizing population structures. A phylogenetic tree for 284 strains showed that all sequenced strains consisted of 5 clades: T1-1 (sequence type [ST]1), T1-2 (mainly ST3), T1-3 (ST17), T2-1 (mainly ST2), and T2-2 (mainly ST14). We identified a putative recombination block containing 6 genes (MPN366‒371). Macrolide resistance involving 23S rRNA mutations was detected for each clade. Clonal expansion of macrolide resistance occurred mostly within subtype 1 strains, of which clade T1-2 showed the highest recombination rate and genome diversity. Functional characterization of recombined regions provided clarification of the biologic role of these recombination events in the evolution of M. pneumoniae.

Nuclear accumulation of KPNA2 impacts radioresistance through positive regulation of the PLSCR1-STAT1 loop in lung adenocarcinoma.

Lung adenocarcinoma (ADC) is the predominant histological type of lung cancer, and radiotherapy is one of the current therapeutic strategies for lung cancer treatment. Unfortunately, biological complexity and cancer heterogeneity contribute to radioresistance development. Karyopherin α2 (KPNA2) is a member of the importin α family that mediates the nucleocytoplasmic transport of cargo proteins. KPNA2 overexpression is observed across cancer tissues of diverse origins. However, the role of KPNA2 in lung cancer radioresistance is unclear. Herein, we demonstrated that high expression of KPNA2 is positively correlated with radioresistance and cancer stem cell (CSC) properties in lung ADC cells. Radioresistant cells exhibited nuclear accumulation of KPNA2 and its cargos (OCT4 and c-MYC). Additionally, KPNA2 knockdown regulated CSC-related gene expression in radioresistant cells. Next-generation sequencing and bioinformatic analysis revealed that STAT1 activation and nuclear phospholipid scramblase 1 (PLSCR1) are involved in KPNA2-mediated radioresistance. Endogenous PLSCR1 interacting with KPNA2 and PLSCR1 knockdown suppressed the radioresistance induced by KPNA2 expression. Both STAT1 and PLSCR1 were found to be positively correlated with dysregulated KPNA2 in radioresistant cells and ADC tissues. We further demonstrated a potential positive feedback loop between PLSCR1 and STAT1 in radioresistant cells, and this PLSCR1-STAT1 loop modulates CSC characteristics. In addition, AKT1 knockdown attenuated the nuclear accumulation of KPNA2 in radioresistant lung cancer cells. Our results collectively support a mechanistic understanding of a novel role for KPNA2 in promoting radioresistance in lung ADC cells.

Genomic Insight into the Spread of Meropenem-Resistant Streptococcus pneumoniae Spain23F-ST81, Taiwan.

Incidence of invasive pneumococcal disease caused by antimicrobial-resistant Streptococcus pneumoniae types not included in pneumococcal conjugate vaccines has increased, including a penicillin- and meropenem-resistant serotype 15A-ST63 clone in Japan. During 2013-2017, we collected 206 invasive pneumococcal isolates in Taiwan for penicillin and meropenem susceptibility testing. We found serotypes 15B/C-ST83 and 15A-ST63 were the most prevalent penicillin- and meropenem-resistant clones. A transformation study confirmed that penicillin-binding protein (PBP) 2b was the primary meropenem resistance determinant, and PBP1a was essential for high-level resistance. The rate of serotype 15B/C-ST83 increased during the study. All 15B/C-ST83 isolates showed an ermB macrolide resistance genotype. Prediction analysis of recombination sites revealed 12 recombination regions in 15B/C-ST83 compared with the S. pneumoniae Spain23F-ST81 genome. Pneumococcal clones rapidly recombine to acquire survival advantages and undergo local expansion under the selective pressure exerted by vaccines and antimicrobial drugs. The spread of 15B/C-ST83 is alarming for countries with high antimicrobial pressure.

Proteomic characterization of arsenic and cadmium exposure in bladder cells.

RATIONALE: Accumulating evidence has linked prolonged exposure to heavy metals to cancer occurrence in the urinary system. However, the specific biological mechanisms responsible for the association of heavy metals with the unusually high incidence of upper tract urothelial carcinoma in Taiwan are complex and incompletely understood. METHODS: To elucidate the specific biological mechanism and identify molecular indicators of the unusually high association of upper tract urothelial carcinoma with heavy metal exposure, protein expression following the treatment of T24 human bladder carcinoma and RT4 human bladder papilloma cell line models with arsenic (As) and cadmium (Cd) was studied. Proteomic changes in these cell models were integrated with data from a human bladder cancer (BLCA) tissue proteome to identify possible protein indicators of heavy metal exposure. RESULTS: After mass spectrometry based proteomic analysis and verification by Western blotting procedures, we identified 66 proteins that were up-regulated and 92 proteins that were down-regulated in RT4 cell extracts after treatment with As or Cd. Some 52 proteins were up-regulated and 136 proteins were down-regulated in T24 cell extracts after treatment with Cd. We further confirmed that down-expression of the PML (promyelocytic leukemia) protein was sustained for at least 75 days after exposure of bladder cells to As. Dysregulation of these cellular proteins by As was associated with three biological pathways. Immunohistochemical analyses of paraffin-embedded BLCA tissue slides confirmed that PML protein expression was decreased in BLCA tumor cells compared with adjacent noncancerous epithelial cells. CONCLUSIONS: These data suggest that PML may play an important role in the pathogenesis of BLCA and may be an indicator of heavy metal exposure in bladder cells.

Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan.

Most cases of oral squamous cell carcinoma (OSCC) develop from visible oral potentially malignant disorders (OPMDs). The latter exhibit heterogeneous subtypes with different transformation potentials, complicating the early detection of OSCC during routine visual oral cancer screenings. To develop clinically applicable biomarkers, we collected saliva samples from 96 healthy controls, 103 low-risk OPMDs, 130 high-risk OPMDs, and 131 OSCC subjects. These individuals were enrolled in Taiwan's Oral Cancer Screening Program. We identified 302 protein biomarkers reported in the literature and/or through in-house studies and prioritized 49 proteins for quantification in the saliva samples using multiple reaction monitoring-MS. Twenty-eight proteins were successfully quantified with high confidence. The quantification data from non-OSCC subjects (healthy controls + low-risk OPMDs) and OSCC subjects in the training set were subjected to classification and regression tree analyses, through which we generated a four-protein panel consisting of MMP1, KNG1, ANXA2, and HSPA5. A risk-score scheme was established, and the panel showed high sensitivity (87.5%) and specificity (80.5%) in the test set to distinguish OSCC samples from non-OSCC samples. The risk score >0.4 detected 84% (42/50) of the stage I OSCCs and a significant portion (42%) of the high-risk OPMDs. Moreover, among 88 high-risk OPMD patients with available follow-up results, 18 developed OSCC within 5 y; of them, 77.8% (14/18) had risk scores >0.4. Our four-protein panel may therefore offer a clinically effective tool for detecting OSCC and monitoring high-risk OPMDs through a readily available biofluid.

Golgi tethering factor golgin-97 suppresses breast cancer cell invasiveness by modulating NF-κB activity.

BACKGROUND: Golgin-97 is a tethering factor in the trans-Golgi network (TGN) and is crucial for vesicular trafficking and maintaining cell polarity. However, the significance of golgin-97 in human diseases such as cancer remains unclear. METHODS: We searched for a potential role of golgin-97 in cancers using Kaplan-Meier Plotter ( http://kmplot.com ) and Oncomine ( www.oncomine.org ) datasets. Specific functions of golgin-97 in migration and invasion were examined in golgin-97-knockdown and golgin-97-overexpressing cells. cDNA microarray, pathway analysis and qPCR were used to identify gene profiles regulated by golgin-97. The role of golgin-97 in NF-κB signaling pathway was examined by using subcellular fractionation, luciferase reporter assay, western blot analysis and immunofluorescence assay (IFA). RESULTS: We found that low expression of golgin-97 correlated with poor overall survival of cancer patients and was associated with invasiveness in breast cancer cells. Golgin-97 knockdown promoted cell migration and invasion, whereas re-expression of golgin-97 restored the above phenotypes in breast cancer cells. Microarray and pathway analyses revealed that golgin-97 knockdown induced the expression of several invasion-promoting genes that were transcriptionally regulated by NF-κB p65. Mechanistically, golgin-97 knockdown significantly reduced IκBα protein levels and activated NF-κB, whereas neither IκBα levels nor NF-κB activity was changed in TGN46- or GCC185-knockdown cells. Conversely, golgin-97 overexpression suppressed NF-κB activity and restored the levels of IκBα in golgin-97-knockdown cells. Interestingly, the results of Golgi-disturbing agent treatment revealed that the loss of Golgi integrity was not involved in the NF-κB activation induced by golgin-97 knockdown. Moreover, both TGN-bound and cytosolic golgin-97 inhibited NF-κB activation, indicating that golgin-97 functions as an NF-κB suppressor regardless of its subcellular localization. CONCLUSION: Our results collectively demonstrate a novel and suppressive role of golgin-97 in cancer invasiveness. We also provide a new avenue for exploring the relationship between the TGN, golgin-97 and NF-κB signaling in tumor progression.

Is the whole greater than the sum of its parts? De novo assembly strategies for bacterial genomes based on paired-end sequencing.

BACKGROUND: Whole genome sequence construction is becoming increasingly feasible because of advances in next generation sequencing (NGS), including increasing throughput and read length. By simply overlapping paired-end reads, we can obtain longer reads with higher accuracy, which can facilitate the assembly process. However, the influences of different library sizes and assembly methods on paired-end sequencing-based de novo assembly remain poorly understood. RESULTS: We used 250 bp Illumina Miseq paired-end reads of different library sizes generated from genomic DNA from Escherichia coli DH1 and Streptococcus parasanguinis FW213 to compare the assembly results of different library sizes and assembly approaches. Our data indicate that overlapping paired-end reads can increase read accuracy but sometimes cause insertion or deletions. Regarding genome assembly, merged reads only outcompete original paired-end reads when coverage depth is low, and larger libraries tend to yield better assembly results. These results imply that distance information is the most critical factor during assembly. Our results also indicate that when depth is sufficiently high, assembly from subsets can sometimes produce better results. CONCLUSIONS: In summary, this study provides systematic evaluations of de novo assembly from paired end sequencing data. Among the assembly strategies, we find that overlapping paired-end reads is not always beneficial for bacteria genome assembly and should be avoided or used with caution especially for genomes containing high fraction of repetitive sequences. Because increasing numbers of projects aim at bacteria genome sequencing, our study provides valuable suggestions for the field of genomic sequence construction.

[Everolimus combined with all-trans retinoid acid reverses drug resistance in acute promyelocytic leukemia NB4-R1 cells].

OBJECTIVE: To investigate the effect of everolimus(RAD001)combined with all-trans retinoid acid(ATRA) on drug resistance of ATRA-resistance acute promyelocytic leukemia(APL) cell line NB4-R1 and its molecular mechanism. METHODS: APL NB4-R1 cells were treated with different concentrations of RAD001(1 nmol/L, 10 nmol/L and 100 nmol/L) with ATRA(1µmol/L) for 24, 48 and 72 h, respectively. The differentiation of NB4-R1 cells was analyzed by flow cytometry with CD11b staining and nitro blue tetrozolium(NBT) reduction test. Cell cycle was detected by cell cycle staining kit and apoptosis was detected by flow cytometry with Annexin V/PI staining. Protein expressions of LC-3II, PML-RARα, P-P70S6K and P-4E-BP1 were determined by Western blotting. RESULTS: RAD001 combined with ATRA significantly induced NB4-R1 cells differentiation, but RAD001 or ATRA alone did not enhance NB4-R1 differentiation. The co-treatment induced accumulation of cells in G1 phase and decreased the proportion of cells in S phase. The combined treatment had no effect on cell apoptosis. The differentiation rate of NB4-R1 cells in 100 nmol/L RAD001, 1µmol/L ATRA, RAD001 combined with ATRA and control groups was(2.29±0.57)%,(17.06±2.65)%,(54.47±4.91)% and(2.54±0.53)%, respectively; the proportion of cells in G1 phase was(35.20±11.97)%,(33.54±6.25)%,(53.70±8.73)% and(27.40±6.01)%, respectively; cells apoptosis rate was(2.30±0.14)%,(2.25±0.21)%,(2.40±0.28)% and(1.95±0.07)%, respectively. The combination of RAD001 with ATRA significantly inhibited mTOR signaling downstream proteins P-P70S6K, P-4E-BP1 and enhanced autophagy-related protein LC3-II and Beclin 1. The co-treatment also induced degradation of fusion protein PML-RARα. CONCLUSION: RAD001 combined with ATRA can induce cell differentiation, inhibit cell cycle, resulting the reverse of drug resistance in NB4-R1 cells, which is associated with increase of autophagy level and degradation of PML-RARα.

Clinical validation of a saliva-based matrix metalloproteinase-1 rapid strip test for detection of oral cavity cancer.

BACKGROUND: We previously identified matrix metalloproteinase-1 (MMP-1) as one of the most promising salivary biomarkers for oral squamous cell carcinoma (OSCC) and developed a sensitive ELISA for MMP-1 with good performance in detection of OSCC using a cohort of 1160 saliva samples. METHODS: A time-saving rapid strip test (RST) for MMP-1 was developed in this study and its diagnostic performance compared with ELISA using saliva samples from a new cohort of 603 subjects (171 healthy controls, 236 patients with oral potentially malignant disorders, and 196 OSCC patients). RESULTS: Salivary MMP-1 levels measured using RST and ELISA were highly comparable and both assays could effectively distinguish between OSCC and non-cancerous groups. Similar to ELISA, receiver operating characteristic curve analysis of the MMP-1 RST was effective in identifying patients with OSCC at different oral cavity sites and stages. CONCLUSIONS: Salivary MMP-1 can be sensitively detected using both RST and ELISA methods. Our newly developed point-of-care MMP-1 RST is a promising in vitro diagnostic device (IVD) that may serve as a novel auxiliary tool in the routine clinical detection and monitoring of OSCC.

Sequential use of capsular typing and whole-genome sequencing-based analysis for transmission of carbapenem-resistant Acinetobacter baumannii in a tertiary medical center.

BACKGROUND: During the COVID-19 pandemic, there has been an increasing trend in healthcare-associated infections (HAIs) caused by carbapenem-resistant Acinetobacter baumannii (CRAB), posting a global public health concern. The heightened sensitivity of whole-genome sequencing (WGS) renders it an optimal and potent tool for monitoring outbreaks and tracing the transmission routes of nosocomial pathogens. METHOD: We collected CRAB isolates from March 1, 2023, to April 6, 2023 in Chang Gung Memorial Hospital Lin Kou branch, a tertiary medical center in northern Taiwan. Any two or more isolates with the same identifiable capsular K-locus (KL) types were selected, and analyzed via WGS to identify putative transmission clusters, combined with epidemiologic and retrospective analysis on medical records to confirm risk factors and hidden transmission chains. RESULT: A total of 48 non-redundant CRAB isolates were collected, belonging to ST2 of Pasteur MLST scheme and identifiable KL types of KL2, KL3, KL9, KL10, KL22, KL52. Excluding the KL types that was only found in 1 case, KL2 (n = 9, 22.5 %), KL3 (n = 24, 60 %), KL9 (n = 3, 7.5 %), and KL10 (n = 4, 10 %) were selected for further WGS analysis. Four distinct transmission clusters comprised of 2, 3, 10, and 23 cases were identified on a basis of phylogenetic status. 12 probable transmission chains were revealed, and 2 hidden transmission routes can be speculated. CONCLUSION: This study referred to some hidden transmission chains that may be missed from traditional surveillance measures. Despite its low prevalence and high cost currently, implementing WGS could be a efficient, prompt, and unequivocal option for future MDRO infection control.

Genomic analysis of penicillin-binding proteins and recombination events in an emerging amoxicillin- and meropenem-resistant PMEN3 (Spain9V-3, ST156) variant in Taiwan and comparison with global descendants of this lineage.

From 2008 to 2020, the Taiwan National Notifiable Disease Surveillance System database demonstrated that the incidence of non-vaccine serotype 23A invasive pneumococcal disease (IPD) approximately doubled. In this study, 276 non-repetitive pneumococcal clinical isolates were collected from two medical centers in Taiwan between 2019 and 2021. Of these 267 pneumococci, 60 were serotype 23A. Among them, 50 (83%) of serotype 23A isolates belonged to the sequence type (ST) 166 variant of the Spain9V-3 clone. Pneumococcal 23A-ST166 isolates were collected to assess their evolutionary relationships using whole-genome sequencing. All 23A-ST166 isolates were resistant to amoxicillin and meropenem, and 96% harbored a novel combination of penicillin-binding proteins (PBPs) (1a:2b:2x):15:11:299, the newly identified PBP2x-299 in Taiwan. Transformation of the pbp1a, pbp2b, and pbp2x alleles into the ß-lactam-susceptible R6 strain revealed that PBP2x-299 and PBP2b-11 increased the MIC of ceftriaxone and meropenem by 16-fold, respectively. Prediction analysis of recombination sites in PMEN3 descendants (23A-ST166 in Taiwan, 35B-ST156 in the United States, and 11A-ST838/ST6521 in Europe) showed that adaptive evolution involved repeated, selectively favored convergent recombination in the capsular polysaccharide synthesis region, PBPs, murM, and folP genome sites. In the late 13-valent pneumococcal conjugate vaccine era, PMEN3 continuously displayed an evolutionary capacity for global dissemination and persistence, increasing IPD incidence, leading to an offset in the decrease of pneumococcal conjugate vaccine serotype-related diseases, and contributing to high antibiotic resistance. A clonal shift with a highly ß-lactam-resistant non-vaccine serotype 23A, from ST338 to ST166, increased in Taiwan. ST166 is a single-locus variant of the Spain9V-3 clone, which is also called the PMEN3 lineage. All 23A-ST166 isolates, in this study, were resistant to amoxicillin and meropenem, and 96% harbored a novel combination of penicillin-binding proteins (PBPs) (1a:2b:2x):15:11:299. PBP2x-299 and PBP2b-11 contributed to the increasing MIC of ceftriaxone and meropenem, respectively. Prediction analysis of recombination sites in PMEN3 descendants showed that adaptive evolution involved repeated, selectively favored convergent recombination in the capsular polysaccharide synthesis region, PBPs, murM, and folP genome sites. In the late 13-valent pneumococcal conjugate vaccine era, PMEN3 continuously displays the evolutionary capacity for dissemination, leading to an offset in the decrease of pneumococcal conjugate vaccine serotype-related diseases and contributing to high antibiotic resistance.

Prognostic value of an APOBEC3 deletion polymorphism for glioma patients in Taiwan.

OBJECTIVE: The molecular pathogenesis of malignant gliomas, characterized by diverse tumor histology with differential prognosis, remains largely unelucidated. An APOBEC3 deletion polymorphism, with a deletion in APOBEC3B, has been correlated to risk and prognosis in several cancers, but its role in glioma is unclear. The authors aimed to examine the clinical relevance of the APOBEC3 deletion polymorphism to glioma risk and survival in a glioma patient cohort in Taiwan. METHODS: The authors detected deletion genotypes in 403 glioma patients and 1365 healthy individuals in Taiwan and correlated the genotypes with glioma risk, clinicopathological factors, patient survival, and patient sex. APOBEC3 gene family expression was measured and correlated to the germline deletion. A nomogram model was constructed to predict patient survival in glioma. RESULTS: The proportion of APOBEC3B-/- and APOBEC3B+/- genotypes was higher in glioblastoma (GBM) patients than healthy individuals and correlated with higher GBM risk in males. A higher percentage of cases with APOBEC3B- was observed in male than female glioma patients. The presence of APOBEC3B-/- was correlated with better overall survival (OS) in male astrocytic glioma patients. No significant correlation of the genotypes to glioma risk and survival was observed in the female patient cohort. Lower APOBEC3B expression was observed in astrocytic glioma patients with APOBEC3B-/- and was positively correlated with better OS. A 5-factor nomogram model was constructed based on male patients with astrocytic gliomas in the study cohort and worked efficiently for predicting patient OS. CONCLUSIONS: The germline APOBEC3 deletion was associated with increased GBM risk and better OS in astrocytic glioma patients in the Taiwan male population. The APOBEC3B deletion homozygote was a potential independent prognostic factor predicting better survival in male astrocytic glioma patients.

T4-Like genome organization of the Escherichia coli O157:H7 lytic phage AR1.

We report the genome organization and analysis of the first completely sequenced T4-like phage, AR1, of Escherichia coli O157:H7. Unlike most of the other sequenced phages of O157:H7, which belong to the temperate Podoviridae and Siphoviridae families, AR1 is a T4-like phage known to efficiently infect this pathogenic bacterial strain. The 167,435-bp AR1 genome is currently the largest among all the sequenced E. coli O157:H7 phages. It carries a total of 281 potential open reading frames (ORFs) and 10 putative tRNA genes. Of these, 126 predicted proteins could be classified into six viral orthologous group categories, with at least 18 proteins of the structural protein category having been detected by tandem mass spectrometry. Comparative genomic analysis of AR1 and four other completely sequenced T4-like genomes (RB32, RB69, T4, and JS98) indicated that they share a well-organized and highly conserved core genome, particularly in the regions encoding DNA replication and virion structural proteins. The major diverse features between these phages include the modules of distal tail fibers and the types and numbers of internal proteins, tRNA genes, and mobile elements. Codon usage analysis suggested that the presence of AR1-encoded tRNAs may be relevant to the codon usage of structural proteins. Furthermore, protein sequence analysis of AR1 gp37, a potential receptor binding protein, indicated that eight residues in the C terminus are unique to O157:H7 T4-like phages AR1 and PP01. These residues are known to be located in the T4 receptor recognition domain, and they may contribute to specificity for adsorption to the O157:H7 strain.

Effects of Probiotics on Gut Microbiomes of Extremely Preterm Infants in the Neonatal Intensive Care Unit: A Prospective Cohort Study.

Background: Probiotics have been previously reported to reduce the incidence of necrotizing enterocolitis (NEC) in extremely preterm infants, but the mechanisms by which the probiotics work remain unknown. We aimed to investigate the effects of probiotics on the gut microbiota of extremely preterm infants. Methods: A prospective cohort study was conducted on 120 extremely preterm neonates (gestational age ≤ 28 weeks) between August 2019 and December 2021. All neonates were divided into the study (receiving probiotics) and the control (no probiotics) groups. Multivariate logistic regression analysis was performed to investigate the significantly different compositions of gut microbiota between these two groups. The effects of probiotics on the occurrence of NEC and late-onset sepsis were also investigated. Results: An increased abundance of Lactobacillus was noted in neonates who received the probiotics (AOR 4.33; 95% CI, 1.89-9.96, p = 0.009) when compared with the control group. Subjects in the probiotic group had significantly fewer days of total parenteral nutrition (median [interquartile range, IQR]) 29.0 (26.8-35.0) versus 35.5 (27.8-45.0), p = 0.004) than those in the control group. The probiotic group had a significantly lower rate of late-onset sepsis than the control group (47.1% versus 70.0%, p = 0.015), but the rate of NEC, duration of hospitalization and the final in-hospital mortality rates were comparable between these two groups. Conclusions: Probiotic supplementation of extremely preterm infants soon after the initiation of feeding increased the abundance of Lactobacillus. Probiotics may reduce the risk of late-onset sepsis, but further randomized controlled trials are warranted in the future.

Exploring the association between capsular types, sequence types, and carbapenemase genes in Acinetobacter baumannii.

Acinetobacter baumannii is the main cause of nosocomial infections, which are increasingly difficult to treat due to the emergence of carbapenem resistance. This study focused on major carbapenemase genes and explored the association between carbapenemase genes, sequence types (ST types), and capsular types (K types). A total of 98 carbapenem-resistant A. baumannii (CRAB) strains were collected from two hospitals, the Chang Gung Memorial Hospital-Lin Kou branch (LCGMH) in northern Taiwan and the CGMH-Kaohsiung branch (KCGMH) in southern Taiwan, from 2015 to 2017. Major carbapenemase genes of class A, B, and D ß-lactamases were detected by polymerase chain reaction. All strains except 1 were positive for blaOXA-51-like, 76 strains (77.6%) carried blaOXA-23-like, and 25 strains (25.5%) carried blaOXA-24-like. The regional distribution showed that blaOXA-23-like was more common than blaOXA-24-like in both hospitals (85.3% and 60% in LCGMH and KCGMH, respectively); however, the percentage of blaOXA-24-like was much higher in KCGMH (46.7%) than in LCGMH (16.2%). Oxford multilocus sequence typing and global optimal eBURST analysis were conducted for 59 strains. The results of this study showed the association between blaOXA gene patterns, ST types, and K types and demonstrated that four major K types, KL2, KL10, KL22, and KL52, which were associated with specific ST types, were mainly clustered into clonal complexes CC208 and CC549 (a unique clonal complex found in Taiwan). These findings provide important information for monitoring the epidemiology and dissemination of this pathogen.

Machine Learning Algorithms to Predict Mortality of Neonates on Mechanical Intubation for Respiratory Failure.

BACKGROUND: Early identification of critically ill neonates with poor outcomes can optimize therapeutic strategies. We aimed to examine whether machine learning (ML) methods can improve mortality prediction for neonatal intensive care unit (NICU) patients on intubation for respiratory failure. METHODS: A total of 1734 neonates with respiratory failure were randomly divided into training (70%, n = 1214) and test (30%, n = 520) sets. The primary outcome was the probability of NICU mortality. The areas under the receiver operating characteristic curves (AUCs) of several ML algorithms were compared with those of the conventional neonatal illness severity scoring systems including the NTISS and SNAPPE-II. RESULTS: For NICU mortality, the random forest (RF) model showed the highest AUC (0.939 (0.921-0.958)) for the prediction of neonates with respiratory failure, and the bagged classification and regression tree model demonstrated the next best results (0.915 (0.891-0.939)). The AUCs of both models were significantly better than the traditional NTISS (0.836 (0.800-0.871)) and SNAPPE-II scores (0.805 (0.766-0.843)). The superior performances were confirmed by higher accuracy and F1 score and better calibration, and the superior and net benefit was confirmed by decision curve analysis. In addition, Shapley additive explanation (SHAP) values were utilized to explain the RF prediction model. CONCLUSIONS: Machine learning algorithms increase the accuracy and predictive ability for mortality of neonates with respiratory failure compared with conventional neonatal illness severity scores. The RF model is suitable for clinical use in the NICU, and clinicians can gain insights and have better communication with families in advance.

An Outbreak of tet(X6)-Carrying Tigecycline-Resistant Acinetobacter baumannii Isolates with a New Capsular Type at a Hospital in Taiwan.

Dissemination of multidrug-resistant, particularly tigecycline-resistant, Acinetobacter baumannii is of critical importance, as tigecycline is considered a last-line antibiotic. Acquisition of tet(X), a tigecycline-inactivating enzyme mostly found in strains of animal origin, imparts tigecycline resistance to A. baumannii. Herein, we investigated the presence of tet(X) variants among 228 tigecycline-non-susceptible A. baumannii isolates from patients at a Taiwanese hospital via polymerase chain reaction using a newly designed universal primer pair. Seven strains (3%) carrying tet(X)-like genes were subjected to whole genome sequencing, revealing high DNA identity. Phylogenetic analysis based on the PFGE profile clustered the seven strains in a clade, which were thus considered outbreak strains. These strains, which were found to co-harbor the chromosome-encoded tet(X6) and the plasmid-encoded blaOXA-72 genes, showed a distinct genotype with an uncommon sequence type (Oxford ST793/Pasteur ST723) and a new capsular type (KL129). In conclusion, we identified an outbreak clone co-carrying tet(X6) and blaOXA-72 among a group of clinical A. baumannii isolates in Taiwan. To the best of our knowledge, this is the first description of tet(X6) in humans and the first report of a tet(X)-like gene in Taiwan. These findings identify the risk for the spread of tet(X6)-carrying tigecycline- and carbapenem-resistant A. baumannii in human healthcare settings.

Clonal spread of macrolide-resistant Mycoplasma pneumoniae sequence type-3 and type-17 with recombination on non-P1 adhesin among children in Taiwan.

OBJECTIVES: Mycoplasma pneumoniae is currently the most commonly detected bacterial cause of childhood community-acquired pneumonia in several countries. Of note, clonal expansion of macrolide-resistant ST3 occurred in Japan and South Korea. An alarming surge in macrolide resistance complicates the treatment of pneumonia. We aimed to evaluate the clinical manifestation and clonal relatedness of M. pneumoniae circulating among children in Taiwan. METHODS: We prospectively enrolled 626 children with radiologically confirmed pneumonia between 2017 and 2019. An M. pneumoniae infection was suspected on clinical grounds, and tested by real-time PCR and oropharyngeal swab cultures. We used multilocus sequence typing and whole-genome sequencing to characterize the genetic features of M. pneumoniae. RESULTS: A total of 226 children with M. pneumoniae pneumonia were enrolled. Macrolide resistance was found in 77% (174/226) of patients. Multi-locus sequence typing revealed that ST3 (n = 93) and its single-locus variant ST17 (n = 84) were the predominant clones among macrolide-resistant strains. ST17 presented clinical characteristics comparable to its ancestor ST3. On multivariate analysis, macrolide resistance (OR 3.5; 95% CI 1.4-8.5; p 0.007) was independently associated with fever >72 hours after macrolide treatment. By whole-genome sequencing, prediction analysis of recombination sites revealed one recombination site in ST3 and ST17 compared with M29 (a macrolide-sensitive ST3 strain isolated from China in 2005) containing cytadhesin MgpC-like protein, RepMP4 and RepMP5. ST17 had another recombination site containing an adhesin and RepMP2/3. CONCLUSIONS: In addition to macrolide resistance, ST3 and its ST17 variant might evolve through recombination between repetitive sequences and non-P1 cytadhesins for persistent circulation in Taiwan.

ASC modulates HIF-1α stability and induces cell mobility in OSCC.

High-level expression of ASC (Apoptosis-associated speck-like protein containing a CARD) leads to lymph node metastasis in OSCC, but the underlying mechanism remains unclear. Here, we show that HIF-1α participates in ASC-induced metastasis. We identified 195 cell-motion-associated genes that were highly activated in ASC-overexpressed SAS_ASC cells; of them, 14 representative genes were found to be overexpressed in OSCC tissues in our previously reported RNA-seq dataset, OSCC-Taiwan. Nine of the 14 genes were also upregulated in OSCC-TCGA samples. Among the nine genes, RRAS2, PDGFA, and VEGFA, were correlated with poor overall survival of patients in OSCC-TCGA dataset. We further demonstrated that the promoters of these 14 ASC-induced genes contained binding motifs for the transcription-regulating factor, HIF-1α. We observed that ASC interacted with and stabilized HIF-1α in both the cytoplasm and the nucleus under normoxia. Molecules involved in the HIF-1α pathway, such as VHL and PHD2, showed no difference in their gene and protein levels in the presence or absence of ASC, but the expression of HIF-1α-OH, and the ubiquitination of HIF-1α were both decreased in SAS_ASC cells versus SAS_con cells. The migration and invasion activities of SAS_ASC cells were reduced when cells were treated with the HIF-1α synthesis inhibitor, digoxin. Taken together, our results demonstrate that the novel ASC-HIF-1α regulatory pathway contributes to lymph node metastasis in OSCC, potentially suggesting a new treatment strategy for OSCC.