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Intratumour heterogeneity (ITH) has become an important focus of cancer research in recent years. ITH describes the cellular variation that enables tumour evolution, including tumour progression, metastasis and resistance to treatment. The selection and expansion of genetically distinct treatment-resistant cancer cell clones provides one explanation for treatment failure. However, tumour cell variation need not be genetically encoded. In pancreatic ductal adenocarcinoma (PDAC) in particular, the complex tumour microenvironment as well as crosstalk between tumour and stromal cells result in exceptionally variable tumour cell phenotypes that are also highly adaptable. In this review we discuss four different types of phenotypic heterogeneity within PDAC, from morphological to metabolic heterogeneity. We suggest that these different types of ITH are not independent, but, rather, can inform one another. Lastly, we highlight recent findings that suggest how therapeutic efforts may halt PDAC progression by constraining cellular heterogeneity.

Amyloid-β (Aβ) peptides and hyperphosphorylated tau protein are the most important pathological markers of Alzheimer’s disease (AD). Neuroinflammation and oxidative stress are also involved in the development and pathological mechanism of AD. Hypoxia inducible factor-1α (HIF-1α) is a transcriptional factor responsible for cellular and tissue adaption to low oxygen tension. Emerging evidence has revealed HIF-1α as a potential medicinal target for neurodegenerative diseases. On the one hand, HIF-1α increases AβPP processing and Aβ generation by promoting β/γ-secretases and suppressing α-secretases, inactivates microglia and reduces their activity, contributes to microglia death and neuroinflammation, which promotes AD pathogenesis. On the other hand, HIF-1α could resist the toxic effect of Aβ, inhibits tau hyperphosphorylation and promotes microglial activation. In summary, this review focuses on the potential complex roles and the future perspectives of HIF-1α in AD, in order to provide references for seeking new drug targets and treatment methods for AD.

Atrazine, a common chemical pesticide, has toxicity to adult and juvenile amphibians in natural ecosystems; however, it is more common to study its effects on larvae instead of adults. This study assessed the impacts of atrazine in water through short-term exposure (7 days) on male black spotted frog (Pelophylax nigromaculatus) adults fed every day. The jumping ability, including jumping height, distance, time, and speed, was measured by 3D motion analysis software, and the intestinal content microbiota was determined by 16S rRNA amplicon sequencing with QIIME software. The results showed that male P. nigromaculatus exposure to 200 and 500 μg/L atrazine significantly increased jumping distance and jumping time compared to control groups. Conversely, 500 μg/L atrazine treatments significantly decreased the diversity and changed the composition and structure of intestinal content microflora in male P. nigromaculatus compared to control groups. At the phylum level, Chlamydiae was only detected in the control group, and Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria were the dominant microflora in the atrazine treatment groups. At the genus level, the abundance of Lactobacillus and Weissella significantly increased in atrazine treatment groups compared to control groups. This study can provide a new framework based on movement behavior and intestinal microbiota to evaluate the response of amphibians to short-term exposure to environmental pollution.

Jumping ability determines the likelihood of survival and reproduction of amphibians. To study the toxic effect of lead (Pb) on the jumping ability of amphibians, Pelophylax nigromaculata were treated with 0, 1 and 10 mg/L Pb for 10 days and 20 days each. The results showed that for both treatment time (10 days and 20 days), compared to the control group, the 1 mg/L Pb treatment had no significant effect on jumping parameters. Compared to the control group, the maximum jumping height, distance, speed and acceleration in the 10-day 10 mg/L Pb treatment group increased significantly, but after 20 days the jumping parameters decreased significantly. With an increase in duration and concentration, the correlation coefficient and the correlation between these jumping parameters decreased. Our results indicate that an exposure to 10 mg/L Pb for 20 days has a significant negative effect on the jumping ability of amphibians.

Background: Pathogenic factors embedded in the germline genome are widely recognized as being crucial to breast cancer development. However, current knowledge is either concentrated on the pathogenic variants of a few individual genes or SNPs distributed sparsely across the genome in non-coding regions. Methods: We developed a multi-layered framework, DAGG, which converts somatic mutations or germline rare coding variants (gRCVs) into a functional spectrum of dozens of cellular functions and signaling pathways to identify potential pathogenic factors.Findings: We analyzed whole-exome sequencing (WES) data of 726 germline DNA samples and 169 breast tumor DNA samples from breast cancer patients with various pathological types and cancer-free female subjects, we found that germline pathogens of breast cancers were (1) mainly distributed in HER2-negative subtypes, and (2) involved Her2 signaling pathway activation and immune suppression. These computational discoveries were experimentally validated and can provide digital features to explain the germline differences between diseased and healthy genome (AUC = 0.76). Furthermore, an individual’s risk for breast cancer can be estimated by calculating the combined effects of these identified germline pathogens. Carriers of BRCA1/2 pathogenic variants were found to have a significantly higher average risk (p = 0.02). Interpretation: The results demonstrated that the identified pathogenic mechanisms by DAGG were compatible with our current understanding of the causes of breast cancer. Moreover, DAGG provides improved performance over currently used polygenic risk score method of measuring complex disease risks. Our framework promises possible future applications for the prevention, diagnosis, and treatment of breast cancer. Citation Format: Mei Yang, Yanhui Fan, Zhi-Yong Wu, Zhendong Feng, Qiangzu Zhang, Shunhua Han, Zhonghai Zhang, Xu Li, Yiqing Xue, Xiaoling Li, Meixia Hu, Jieqing Li, Weiping Li, Hongfei Gao, Ciqiu Yang, Chunming Zhang, Liulu Zhang, Teng Zhu, Minyi Cheng, Fei Ji, Juntao Xu, Hening Cui, Guangming Tan, Michael Q Zhang, Changhong Liang, Zaiyi Liu, You-Qiang Song, Gang Niu, Kun Wang. Rare variants in the germline genome holistically determine receptor-independent Her2 signaling pathway activation and immune suppression, shaping pathological type and risk of HER2-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-29.

Context Polygonatum sibiricum polysaccharide (PSP), derived from Polygonatum sibiricum Delar. ex Redoute (Liliaceae), is known to be able to delay the ageing process. However, the specific mechanisms underlying these effects are not clear. Objective To investigate the mechanisms underlying the effects of PSP treatment on brain ageing by the application of transcriptomic analysis. Materials and methods Forty Kunming mice were randomly divided into four groups (control, d-galactose, low-dose PSP, high-dose PSP). Mice were administered d-galactose (50 mg/kg, hypodermic injection) and PSP (200 or 400 mg/kg, intragastric administration) daily for 60 days. Behavioural responses were evaluated with the Morris water maze and the profiles of circRNA, miRNA, and mRNA, in the brains of experimental mice were investigated during the ageing process with and without PSP treatment. Results PSP improved cognitive function during brain ageing, as evidenced by a reduced escape latency time (p

BackgroundReprogramming somatic cells to induced pluripotent stem cells (iPSCs) has opened new therapeutic possibilities. However, karyotypic abnormalities detected in iPSCs compromised their utility, especially chromosomal aberrations found at early passages raised serious safety concerns. The mechanism underlying the chromosomal abnormality in early-passage iPSCs is not known.MethodsHuman dermal fibroblasts (HDFs) were stimulated with KMOS (KLF4, cMYC, OCT4 and SOX2) proteins to enhance their proliferative capacity and many vigorous clones were obtained. Clonal reprogramming was carried out by KMOS mRNAs transfection to confirm the ‘chromosomal mutagenicity’ of reprogramming process. Subculturing was performed to examine karyotypic stability of iPSCs after the re-establishment of stemness. And antioxidant N-acetyl-cysteine (NAC) was added to the culture medium for further confirmming the mutagenicity in the first few days of reprogramming.ResultsChromosomal aberrations were found in a small percentage of newly induced iPS clones by reprogramming transcription factors. Clonal reprogramming ruled out the aberrant chromosomes inherited from rare karyotypically abnormal parental cell subpopulation. More importantly, the antioxidant NAC effectively reduced the occurrence of chromosomal aberrations at the early stage of reprogramming. Once iPS cell lines were established, they restored karyotypic stability in subsequent subculturing.ConclusionsOur results provided the first line of evidence for the ‘chromosomal mutagenicity’ of reprogramming process.

In the developing spinal cord, the majority of oligodendrocyte progenitor cells (OPCs) are induced in the ventral neuroepithelium under the control of the Sonic Hedgehog (Shh) signaling pathway, whereas a small subset of OPCs are generated from the dorsal neuroepithelial cells independent of the Shh pathway. Although dorsally-derived OPCs (dOPCs) have been shown to participate in local axonal myelination in the dorsolateral regions during development, it is not known whether they are capable of migrating into the ventral region and myelinating ventral axons. In this study, we confirmed and extended the previous study on the developmental potential of dOPCs in the absence of ventrally-derived OPCs (vOPCs). In Nestin-Smo conditional knockout (cKO) mice, when ventral oligodendrogenesis was blocked, dOPCs were found to undergo rapid amplification, spread to ventral spinal tissue, and eventually differentiated into myelinating OLs in the ventral white matter with a temporal delay, providing genetic evidence that dOPCs are capable of myelinating ventral axons in the mouse spinal cord. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-020-00593-5) contains supplementary material, which is available to authorized users.

The emergence of carbapenemase-producing Enterobacteriaceae has become a major global concern. OXA-48-like carbapenemase gene and its variants have been increasingly reported worldwide. This study reported the first OXA-181-producing Klebsiella quasipneumoniae isolate in Malaysia. This bacterium was isolated from blood specimen of a three-year-old boy with Alagille syndrome who had liver biopsy on October 2016. He had undergone liver transplant in India ten months previously. The genotypic and phenotypic characteristics of the strain were elucidated in this study. To our best knowledge, this is the first report of OXA-181-producing K. quasipneumoniae in Malaysia.

Oral cancer belongs to the group of head and neck cancers. If not diagnosed or treated early, it can be life threatening. Epithelial-mesenchymal transition (EMT) plays an important role in tumor formation and progression. An increase in the presence of the EMT phenotype causes tumor cell proliferation, migration, invasion, and poor prognosis. Therefore, attenuating carcinogenesis via EMT inhibition is a good strategy. Herein, we will determine the pharmacological effects of chrysophanol on the EMT in FaDu cells. To analyze EMT, we detected the expression EMT markers, includingα-SMA,β-catenin, vimentin, N-cadherin, E-cadherin, phospho-GSK-3β, and nuclear translocations of p65 andβ-catenin by western blotting. Additionally, accumulating evidence indicates that reactive oxygen species (ROS) mediate EMT. Our results showed that the level of ROS was significantly increased after chrysophanol treatment. We further speculated that chrysophanol-mediated EMT and metastasis are involved in the Wnt-3-dependent signaling pathway. The inhibition of the EMT phenotype and metastasis and accumulation of ROS caused by chrysophanol was reversed by treatment with the Wnt-3 agonist Bml 284. Therefore, our findings indicated that chrysophanol altered EMT formation, ROS accumulation, and metastasis via the Wnt-3-dependent signaling pathway.

Temperature is an important microenvironmental factor that functions epigenetically in normal embryonic development. However, the effect of hyperthermia in the stem cells is not fully understood. Oct4 is a tightly regulated master regulator of pluripotency maintenance in stem cells and during early embryonic development. We report here that Oct4 protein level was significantly reduced under hyperthermia in mouse blastocysts and embryonic stem cells. The reduction in Oct4 in the mouse embryonic stem cells under hyperthermia was mediated by a ubiquitin-proteasome pathway that was dependent on the activity of death-associated protein kinase 1 (Dapk1) to phosphorylate its substrate, Pin1. Our results imply that the depletion of Oct4 via brief hyperthermia, such as a high fever, during early pregnancy might severely impair the growth of the mammalian embryo or even cause its death.

Background: CDK 4/6 inhibitors have become frontline treatment for patients (pts) with advanced hormone receptor (HR) positive breast cancer. There is limited data regarding post-progression outcomes in pts receiving CDK 4/6 inhibitors as standard of care. In this study, we analyzed treatment patterns and clinical outcomes of post-progression treatment in pts with advanced HR positive breast cancer after receiving CDK 4/6 inhibitors. Methods: Pts with metastatic breast cancer receiving CDK 4/6 inhibitors who received treatment after progression were identified from a cohort of pts at MDACC. Pts receiving CDK 4/6 inhibitors in a clinical trial were excluded. Clinical and demographic data as well as progression-free survival (PFS) was collected. Data was analyzed using Fischer's exact test for categorized variables and T test/Wilcoxon rank-sum test for continuous variables. PFS was analyzed using the Kaplan Meier method. Results: 206 pts who met eligibility criteria were included. Median age at metastatic diagnosis was 56 years-old and the majority of pts were white (n=160, 77.7%) with a pre- treatment performance status of 0 (n=168, 81.6%). The largest fraction of pts (n=98, 47.6%) received a chemotherapy agent following progression on a CDK 4/6 inhibitor while 19.4% (n=40) received endocrine therapy, 21.8% (n=45) received a biologic, 6.3% (N=13) received a different CDK 4/6 inhibitor and 4.9% (n=10) received an investigational therapy. The majority of pts who progressed on a subsequent agent had visceral progression (n=193, 93.7%). The median time to progression on an agent after CDK 4/6 inhibitor was 122 days. There was no statically significant difference in time to progression after CDK 4/6 inhibitor between pts receiving different classes of therapies, including CDK 4/6 inhibitors and investigational therapies (p=0.3). Conclusion: Pts in our cohort were more likely to receive chemotherapy following progression on a CDK 4/6 inhibitor. Reassuringly, there was no significant difference between time to progression on different agents. As CDK 4/6 inhibitors become frontline therapy for HR positive metastatic breast cancer, it is important to evaluate the sequencing of therapies as well as outcomes on subsequent agents. Citation Format: Katherine Keaney Clifton, Akshara Singareeka Raghavendra, Min Yi, Jennifer Litton, Debu Tripathy, Meghan Karuturi. Practice patterns and outcomes of post progression treatment after CDK 4/6 inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-11-03.

The chemical constituents and the antioxidant, antimicrobial, and cytotoxic activities of fresh rhizome essential oil (FR-EO) and dry rhizome essential oil (DR-EO) of Zingiber zerumbet (L.) Smith obtained from Southwest China were compared. Zerumbone was the predominant component in both FR-EO and DR-EO (75.0% and 41.9%, respectively). FR-EO, DR-EO, and zerumbone were all demonstrated to have significant antimicrobial capacity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Proteus vulgaris, with minimum inhibitory concentration (MIC) ranging from 31.25 to 156.25 μg/mL and minimum bactericidal concentration (MBC) ranging from 62.50 to 625.00 μg/mL. Zerumbone showed the strongest antimicrobial potential against all tested microorganisms compared with the fresh and dry rhizome essential oils. FR-EO was found to be more active than DR-EO against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Proteus vulgaris. FR-EO, DR-EO, and zerumbone all showed significant cytotoxic activity against K562, PC-3, and A549 human tumor cell lines in a time- and concentration-dependent manner. Zerumbone exhibited the strongest antiproliferative activity against all tested human tumor cell lines with an IC50 of 4.21–11.09 μg/mL for 72 h incubation, as compared with the fresh and dry rhizome oils. The cytotoxic activity of FR-EO (IC50: 10.48–14.51 μg/mL for 72 h) was found to be significantly higher (p<0.05) than that of DR-EO (IC50: 13.83–33.24 μg/mL for 72 h). FR-EO, DR-EO, and zerumbone exhibited selective cytotoxic activity to tumor cells, with a significantly low cytotoxicity to normal cells (MRC-5, IC50: 56.98–147.29 μg/mL). However, FR-EO, DR-EO, and zerumbone all exhibited weak free-radical-scavenging activity according to DPPH and ABTS analysis. The findings highlighted in this study show that FR-EO provides appreciably higher content of the bioactive compound, zerumbone, and has higher antimicrobial and cytotoxic properties than DR-EO. Thus, fresh Z. zerumbet rhizome should be preferred in cosmetic, food, and pharmaceutical applications.

Half of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here, we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is required for translation initiation on select transcripts. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated translation occurs on messenger RNAs (mRNAs) with long, structure-prone 5′ leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates main CDS, but not uORF, translation suggesting a role for DAP5 in translation re-initiation. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

Cadmium (Cd) is a non-essential environmental endocrine-disrupting compound found in water and a potential threat to aquatic habitats. Cd has been shown to have various short-term effects on aquatic animals; however, evidence for long-term effects of Cd on vocal communications in amphibians is lacking. To better understand the long-term effects of low-dose Cd on acoustic communication in amphibians, male Xenopus laevis individuals were treated with low Cd concentrations (0.1, 1, and 10 μg/L) via aqueous exposure for 24 months. At the end of the exposure, the acoustic spectrum characteristics of male advertisement calls and male movement behaviors in response to female calls were recorded. The gene and protein expressions of the androgen receptor (AR) were determined using Western blot and RT-PCR. The results showed that long-term Cd treatment affected the spectrogram and formant of the advertisement call. Compared with the control group, 10 μg/L Cd significantly decreased the first and second formant frequency, and the fundamental and main frequency, and increased the third formant frequency. One and 10-μg/L Cd treatments significantly reduced the proportion of individuals responding to female calls and prolonged the time of first movement of the male. Long-term Cd treatment induced a downregulation in the AR protein. Treatments of 0.1, 1, and 10 μg/L Cd significantly decreased the expression of AR mRNA in the brain. These findings indicate that long-term exposure of Cd has negative effects on advertisement calls in male X. laevis.

Studies on the development of central nervous system (CNS) primarily rely on the use of specific molecular markers for different types of neural cells. S100B is widely being used as a specific marker for astrocytes in the CNS. However, the specificity of its expression in astrocyte lineage has not been systematically investigated and thus has remained a lingering issue. In this study, we provide several lines of molecular and genetic evidences that S100B is expressed in both protoplasmic astrocytes and myelinating oligodendrocytes. In the developing spinal cord, S100B is first expressed in the ventral neuroepithelial cells, and later in ALDH1L1+/GS+ astrocytes in the gray matter. Meanwhile, nearly all the S100B+ cells in the white matter are SOX10+/MYRF+ oligodendrocytes. Consistent with this observation, S100B expression is selectively lost in the white matter in Olig2-null mutants in which oligodendrocyte progenitor cells (OPCs) are not produced, and dramatically reduced in Myrf-conditional knockout mutants in which OPCs fail to differentiate. Similar expression patterns of S100B are observed in the developing forebrain. Based on these molecular and genetic studies, we conclude that S100B is not a specific marker for astrocyte lineage; instead, it marks protoplasmic astrocytes in the gray matter and differentiating oligodendrocytes.

The prevalence of type 2 diabetes (T2D) is on the rise worldwide. In addition to genetics and lifestyle factors, exposure to foreign chemical compounds, known as xenobiotics, is emerging as an important risk factor. Pesticides, as a class of xenobiotics, have been credited to significantly increase agricultural production and prevent the spread of vector-borne diseases. However, there is now emerging evidence that long-term pesticide exposure can become toxic, disrupt the body’s balance of energy homeostasis, and contribute to the pathogenesis of many metabolic diseases. If human susceptibility to pesticides results in T2D, looking to other organisms that are immune to pesticides may help solve human disorders. In contrast to incidental exposure on humans, pesticide applications to a detrimental agricultural pest, the two-spotted spider mite, is often futile. The mite’s remarkable ability to detoxify and develop resistance often protects them against energy dysregulation. By studying mites, we learn how they overcome pesticide toxicity, and when translated to humans, this could one day help us overcome and prevent xenobiotic mediated T2D.

Environmental monitoring in public spaces can be used to identify surfaces contaminated by persons with COVID-19 and inform appropriate infection mitigation responses. Research groups have reported detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on surfaces days or weeks after the virus has been deposited, making it difficult to estimate when an infected individual may have shed virus onto a SARS-CoV-2 positive surface, which in turn complicates the process of establishing effective quarantine measures. In this study, we determined that reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of viral RNA from heat-inactivated particles experiences minimal decay over seven days of monitoring on eight out of nine surfaces tested. The properties of the studied surfaces result in RT-qPCR signatures that can be segregated into two material categories, rough and smooth, where smooth surfaces have a lower limit of detection. RT-qPCR signal intensity (average quantification cycle (Cq)) can be correlated to surface viral load using only one linear regression model per material category. The same experiment was performed with infectious viral particles on one surface from each category, with essentially identical results. The stability of RT-qPCR viral signal demonstrates the need to clean monitored surfaces after sampling to establish temporal resolution. Additionally, these findings can be used to minimize the number of materials and time points tested and allow for the use of heat-inactivated viral particles when optimizing environmental monitoring methods.ImportanceEnvironmental monitoring is an important tool for public health surveillance, particularly in settings with low rates of diagnostic testing. Time between sampling public environments, such as hospitals or schools, and notifying stakeholders of the results should be minimal, allowing decisions to be made towards containing outbreaks of coronavirus disease 2019 (COVID-19). The Safer At School Early Alert program (SASEA) [1], a large-scale environmental monitoring effort in elementary school and child care settings, has processed > 13,000 surface samples for SARS-CoV-2, detecting viral signals from 574 samples. However, consecutive detection events necessitated the present study to establish appropriate response practices around persistent viral signals on classroom surfaces. Other research groups and clinical labs developing environmental monitoring methods may need to establish their own correlation between RT - qPCR results and viral load, but this work provides evidence justifying simplified experimental designs, like reduced testing materials and the use of heat-inactivated viral particles.

Background The programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) have shown positive efficacy in several solid cancers due to their targeted antitumour effects. However, the frequency and clinical implication value in prostate cancer still remain unclear. Methods The PD-1/PD-L1 expression was detected by immunohistochemical (IHC) analysis in 96 retrospectively collected cases of prostatic cancer and 44 controls of benign prostatic hyperplasia (BPH). Its correlation with clinicopathological features including age, PSA level, Gleason score, lymph node metastasis, clinical T stage and risk factor grade in prostate cancer was also assessed. Results The PD-L1-positive expression was significantly higher in cancer cases compared with benign tissues, whereas no difference in PD-1 positive expression was found. Moreover, the PD-L1 expression in tumour cells or lymphocytes was associated with Gleason score, but not related to age, preoperative PSA level, clinical T-stage, lymph node metastasis and grade of risk factors. In addition, no association between the positive expression of PD-1 and PD-L1 in tumour cells and lymphocytes was found. Conclusions The expression of PD-L1 not PD-1 is highly prevalent in prostate cancer. PD-L1 is closely related to Gleason score and may be a co-factor associated with the progression of prostate cancer.

Infected aortic aneurysm is a rare but life-threatening condition. Intimal injury, especially in arteries with atherosclerotic plaques, is vulnerable to Salmonella species. In this study, THP-1 macrophage-derived foam cells were used to mimic the atherosclerotic plaques. We investigated the role of different Salmonella serotype infections in foam cell autophagy and inflammatory response. Three different serotypes of Salmonella enterica-Typhimurium, Enteritidis, and Choleraesuis - with or without a virulence plasmid were used in this study. The foam cell model was established and observed under confocal microscopy. The expression of autophagy and inflammasome-related proteins after Salmonella infection was detected by Western blotting, and the secretion of the proinflammatory cytokines interleukin-1β and IL-18 was detected using ELISA. The plasmid-bearing S. enteritidis OU7130- and plasmid-less S. choleraesuis OU7266-infected foam cells both showed a lower expression of beclin-1 at 0.5 h post-infection (hpi) and p62 at 2 hpi as well as a higher expression of caspase-1 at 2 hpi than other infected cells. Moreover, infection of OU7266 induced the highest IL-1β and IL-18 production among the tested strains. In conclusion, the virulence plasmid of S. enteritidis OU7130 induced foam cell inflammatory responses, whereas that of S. choleraesuis played an opposite role. The different serotype of Salmonella may impact the infected aortic aneurysms through regulating inflammatory balance of the macrophage and foam cells in the atherosclerotic plaque.

Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes - Typhimurium, Enteritidis, and Choleraesuis - in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1β, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy - significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1β secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1β secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy. Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes – Typhimurium, Enteritidis, and Choleraesuis – in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1β, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy – significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1β secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1β secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.

Objective: Long non-coding RNAs (lncRNAs) have recently been demonstrated to be involved in craniocerebral disease, but their expression in traumatic brain injury (TBI) is still unearthed. Therefore, we aimed to elucidate the effect of lncRNA CRNDE on TBI. Methods: Firstly, CRNDE expression was determined in serum of TBI patients and healthy controls. The TBI rat model was established based on Feeney's freefall impact method. The modeled rats were injected with siRNA against CRNDE, and the rats' neurobehavioral function were measured. Besides, expression of inflammatory factors, size, shape and number of hippocampal neurons, neuron apoptosis, Beclin I, LC3-I, LC3-II, glial fibrillary acidic protein (GFAP), BrdU, nerve growth factor (NGF), nestin, and neuronal nuclei (NeuN) expression were detected through different methods. Results: In TBI, CRNDE was found to be upregulated. Downregulated CRNDE improved neurobehavioral function, repressed expression of neuroinflammatory factors, elevated number of Nissl bodies, as well as restricted neuronal apoptosis and autophagy in TBI rats. Besides, downregulated CRNDE also promoted expression of GFAP, BrdU, NGF, nestin, and NeuN, thus induced the differentiation of neurons and the directional growth and regeneration of nerve fibers. Conclusion: Altogether, we found that silencing of CRNDE might be able to promote the nerve repair after TBI in rats.

A firm understanding of the genetic relationships among wood properties is a prerequisite for breeding for higher wood quality in Pinus elliottii families. To examine and deal with such relationships, increment cores were sampled at breast height from 1260 trees in 42 open-pollinated families in three 27-year-old Slash pine progeny trials in southern China, and genetic variation, genotype-by-environment (G × E) interaction, genetic correlation and correlated response were investigated. The basic density (BD), dynamic modulus of elasticity (MOED) and tracheid traits were found to be under moderate to strong genetic control for the three locations combined, with individual narrow-sense and family mean heritability ranging from 0.28 to 0.44 and 0.52 to 0.69, respectively. Type B genetic correlation estimates indicated that the G × E interaction had a small-level influence on wood properties. Strong genetic correlations (rg) were found between BD and MOED at the three sites (rg = 0.46–0.85), and BD or MOED showed moderate to strong correlations with most tracheid traits at specific localities. In tree breeding programs, one possible strategy would be to improve pulpwood quality and the strength of structural wood through selection for different wood quality traits.

Salmonella is a common foodborne and zoonotic pathogen. Only a few serovars carry a virulence plasmid (pSV), which enhances the pathogenicity of the host. Here, we investigated the pathogenicity roles of the pSVs among wild-type, plasmid-less, and complemented S. Typhimurium, S. Enteritidis S. Choleraesuis in invasion, phagocytosis, and intracellular bacterial survival in human THP-1 cells and cell death patterns by flow cytometry and difference in cell death patterns between pig and human S. Choleraesuis isolates with large pSCVs. Virulence plasmid (pSTV) led to slightly increasing cellular apoptosis for S. Typhimurium; virulence plasmid (pSEV) enhanced apoptosis and necrosis significantly for S. Enteritidis; and pSCV reduced apoptosis significantly for S. Choleraesuis. After complementation, pSTV increased the intracellular survival of pSCV-less Choleraesuis and the cytotoxicity against human THP-1 cells. Using the Cytochalasin D to differentiate the invasion of S. Choleraaesuis and phagocytosis of THP-1 cells determined that pSCV were responsible for invasion and phagocytosis at 0 h and inhibited intracellular replication in THP-1 cells, and pSTV were responsible for invasion and increased intracellular survival for S. Choleraesuis in THP-1 cells. The human isolates with large pSCV induced more cellular apoptosis and necrosis than the pig isolates. In conclusion, human S. Choleraesuis isolates carrying large pSCVs were more adapted to human THP-1 cells for more cell death than pig isolates with large pSCV. The role of pSVs in invasion, phagocytosis, intracellular survival and apoptosis differed among hosted serovars.

Microorganisms, particularly bacteria, are important components of mature soil capable of high crop yields. However, changes in the diversity and abundance of bacteria during the maturation of parent soil have not been thoroughly elucidated to date. In this study, we determined the diversity of the bacterial community in millet rhizospheres from mellow soil (MS), immature soil (IS), and a mixture of MS and IS (MS-IS mixture) using high-throughput Illumina sequencing. We also analysed soil chemical properties, microbial carbon, microbial nitrogen and enzyme activities, and we correlated them with bacterial taxa. In different rhizosphere soil samples, higher bacterial diversity was found in MS than in IS or the MS-IS mixture. The same trends were observed for soil organic carbon (SOC), available nitrogen (AN), available phosphorus (AP), carbon to nitrogen ratio (C/N), soil microbial carbon, soil microbial nitrogen and soil enzyme activities. Compared to IS, MS harboured a higher abundance of two phyla (Proteobacteria and Actinobacteria) but a lower abundance of five phyla (Chloroflexi, Acidobacteria, Bacteroidetes, Nitrospirae and Firmicutes) and a higher abundance of 22 genera but a lower abundance of 13 genera. A redundancy analysis indicated that the higher abundance of seven genera (Caenimonas, Gemmatimonas, Lysobacter, Ramlibacter, Devosia, Mycobacterium and Streptomyces) was correlated with higher levels of SOC, AN and C/N. Phosphatase, invertase and urease were correlated with higher levels of SOC, AN and C/N. These findings suggest that higher abundance of some specific bacteria accelerates soil maturation by increasing soil chemical properties and soil enzyme activities, thereby leading to higher crop yields.

ObjectivesIn a genome-wide association study, we discovered chromosome 12q15 (defined as rs73329476) as a silica-related pneumoconiosis susceptibility region. However, the causal variants in this region have not yet been reported.MethodsWe systematically screened eight potentially functional single-neucleotide polymorphism (SNPs) in the genes near rs73329476 (carboxypeptidase M (CPM) and cleavage and polyadenylation specific factor 6 (CPSF6)) in a case–control study including 177 cases with silicosis and 204 healthy controls, matched to cases with years of silica dust exposure. We evaluated the associations between these eight SNPs and the development of silicosis. Luciferase reporter gene assays were performed to test the effects of selected SNP on the activity of CPM in the promoter. In addition, a two-stage case–control study was performed to investigate the expression differences of the two genes in peripheral blood leucocytes from a total of 64 cases with silicosis and 64 healthy controls with similar years of silica dust exposure as the cases.ResultsWe found a strong association between the mutant rs12812500 G allele and the susceptibility of silicosis (OR=1.45, 95% CI 1.03 to 2.04, p=0.034), while luciferase reporter gene assays indicated that the mutant G allele of rs12812500 is strongly associated with increased luciferase levels compared with the wild-type C allele (pCPM gene was significantly higher in subjects with silicosis compared with healthy controls.ConclusionsThe rs12812500 variant of the CPM gene may increase silicosis susceptibility by affecting the expression of CPM, which may contribute to silicosis susceptibility with biological plausibility.

Escherichia coli is one major cause of bacterial infections and can horizontally acquire antimicrobial resistance and virulence genes through conjugation. Because conjugative plasmids can rapidly spread among bacteria of different species, the plasmids carrying both antimicrobial resistance and virulence genes may pose a significant threat to public health. Therefore, the identification and characterization of these plasmids may facilitate a better understanding of E. coli pathogenesis and the development of new strategies against E. coli infections. Because iron uptake ability is a potential virulence trait of bacteria, we screened for E. coli conjugative plasmids able to confer both iron uptake ability and ampicillin resistance. The plasmid pEC41, which was derived from the bacteremia clinical isolate EC41, was identified. EC41, which carried the fimH27 allele, belonged to sequence type (ST) 405 and phylogroup D. According to the sequencing analyses, pEC41 was 86 kb in size, and its backbone structure was almost identical to that of another highly conjugative plasmid, pCTX-M3, in which the extended-spectrum β-lactamase gene blaCTX–M–3 was originally identified. pEC41 carried blaCTX–M–3 and blaTEM–1. The ferric citrate uptake (fec) system was identified in pEC41 and was responsible for conferring iron uptake ability. The fec system contributes to the pathogenesis of EC41 in systemic infections but not in urinary tract infections (UTIs). However, this system promoted competitive fitness of a cystitis-associated clinical isolate to colonize urinary tracts. Additionally, the distribution of the fec system was related to E. coli isolates associated with human bacteremia and UTIs. In summary, the present study identified a novel conjugative plasmid, pEC41, which conferred both antimicrobial resistance and an extra iron uptake ability to E. coli. The iron uptake ability was encoded in the fec system and contributed to E. coli pathogenesis. This study is the first to show that the fec system is a virulence factor in E. coli.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a great concern, as carbapenems are the last-line therapy for multidrug-resistant Gram-negative bacteria infections. This study aims to report the epidemiology of CRKP in a teaching hospital in Malaysia based on the molecular genotypic and clinical characteristics of the isolates. Sixty-three CRKP strains were isolated from a tertiary teaching hospital from January 2016 until August 2017. Carbapenemase genes were detected in 55 isolates, with blaOXA-48 (63.5%) as the predominant carbapenemase gene, followed by blaNDM (36.5%). At least one porin loss was detected in nine isolates. Overall, 63 isolates were divided into 30 clusters at similarity of 80% with PFGE analysis. Statistical analysis showed that in-hospital mortality was significantly associated with the usage of central venous catheter, infection or colonization by CRKP, particularly NDM-producers. In comparison, survival analysis using Cox proportional hazards regression identified a higher hazard ratio for patients with a stoma and patients treated with imipenem but a lower hazard ratio for patients with NDM-producing CRKP. OXA-48 carbapenemase gene was the predominant carbapenemase gene in this study. As CRKP infection could lead to a high rate of in-hospital mortality, early detection of the isolates was important to reduce their dissemination.

SummaryHalf of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is selectively required for re-initiation at the main CDS following uORF translation. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated re-initiation occurs on messenger RNAs (mRNAs) with long, structure-prone 5′ leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates re-initiation by unrecycled post-termination 40S subunits. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

Rhynchanthus beesianus is a medicinal, ornamental, and edible plant, and its essential oil has been used as an aromatic stomachic in China. In this study, the chemical constituents, antibacterial, and anti-inflammatory properties of flower essential oil (F-EO), leaf essential oil (L-EO), and stem essential oil (S-EO) of R. beesianus were investigated for the first time. According to the GC-FID/MS assay, the F-EO was mainly composed of bornyl formate (21.7%), 1,8-cineole (21.6%), borneol (9.7%), methyleugenol (7.7%), β-myrcene (5.4%), limonene (4.7%), camphene (4.5%), linalool (3.4%), and α-pinene (3.1%). The predominant components of L-EO were bornyl formate (33.9%), borneol (13.2%), 1,8-cineole (12.1%), methyleugenol (8.0%), camphene (7.8%), bornyl acetate (6.2%), and α-pinene (4.3%). The main components of S-EO were borneol (22.5%), 1,8-cineole (21.3%), methyleugenol (14.6%), bornyl formate (11.6%), and bornyl acetate (3.9%). For the bioactivities, the F-EO, L-EO, and S-EO exhibited significant antibacterial property against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli with the inhibition zones (7.28–9.69 mm), MIC (3.13–12.50 mg/mL), and MBC (6.25–12.50 mg/mL). Besides, the F-EO, L-EO, and S-EO significantly inhibited the production of proinflammatory mediator nitric oxide (NO) (93.15–94.72%) and cytokines interleukin-6 (IL-6) (23.99–77.81%) and tumor necrosis factor-α (TNF-α) (17.69–24.93%) in LPS-stimulated RAW264.7 cells at the dose of 128 μg/mL in the absence of cytotoxicity. Hence, the essential oils of R. beesianus flower, leaf, and stem could be used as natural antibacterial and anti-inflammatory agents with a high application potential in the pharmaceutical and cosmetic fields.

Certain sucrose mono-esters display excellent emulsifying properties as well as a range of other useful physicochemical characteristics that follow from their amphipathic nature. Accordingly, they are playing increasingly important roles in various food manufacturing industries. In this study, an homologous series of 6-O-acylsucrose mono-esters with long and very long fatty acid-derived acyl side-chains has been prepared via enzymatic transesterification. The surface-active properties as well as the antibacterial and antitumor activities of these esters have been studied. Their HLB values were 10.3–15.7 while the mean droplet sizes of the emulsions formed with soybean, colza and olive oils were 380–563 nm. Oil-water emulsions incorporating some of them are more stable, even under conditions of thermal and ionic stress, than those prepared using commercial sucrose esters. Additionally, these esters display activities against Gram-positive bacteria and certain fungi as well as exerting antiproliferative effects on six tumor cell lines. Taken together, these results suggest that the title esters could have considerable utility in the food industry by serving as emulsifiers, as preservatives and even as functional foods.

Hemodialysis requires repeated, reliable access to the systemic circulation; therefore, a well‐functioning vascular access (VA) procedure is crucial for stable hemodialysis. VA infections (VAIs) constitute the most challenging complication and cause considerable morbidity, loss of access, and even death. In this study, we investigated the molecular profiles of different bacterial isolates retrieved from various types of VA grafts. We collected clinical isolates from hemodialysis patients with VAIs in our institution for the period between 2013 and 2018. We identified the bacterial isolates using standard biochemical procedures; we used a polymerase chain reaction for coagulase‐negative staphylococci (CoNS) and Burkholderia cepacia complex (BCC) species identification. The antibiotic resistance and molecular profile were analyzed using the disk diffusion method and multilocus sequence typing, respectively. We studied 150 isolates retrieved from patients with VAI and observed that Staphylococcus aureus was the predominant bacterial species, followed by S. argenteus, BCC, and CoNS. According to multilocus sequence typing data, we identified a wide variety of sequence types (STs) in S. aureus isolates, with ST59, ST45, and ST239 being the predominant types. Burkholderia cepacia with two new ST types, namely ST1723 and ST1724, accounted for most of the BCC infections, along with ST102 B. contaminans, which were mainly isolated from infected tunneled‐cuffed catheters. In summary, the increased incidence of S. argenteus and BCC infections provides insights into their potential clinical effects in VAIs. The various STs identified in different bacterial species indicate the high genetic diversity of bacterial species isolated from VAIs in our institution., Staphylococcus aureus was the predominant pathogen in vascular access infections in our single‐institution study. Burkholderia cepacia complex was exclusively retrieved from tunneled‐cuffed catheter‐related infections. The increased incidence of Staphylococcus argenteus and Burkholderia cepacia complex infections provides insights into their potential clinical effects in vascular access infections.

Regulated expression of genetic elements that either encode polypeptides or various types of functional RNA is a fundamental goal for gene therapy. Inducible expression may be preferred over constitutive promoters to allow clinician-based control of gene expression. Existing Tet-On systems represent one of the tightest rheostats for control of gene expression in mammals. However, basal expression in absence of tetracycline compromises the widespread application of Tet-controlled systems in gene therapy. We demonstrate that the order of P2A-linked genes of interest was critical for maximal response and tightness of a chimeric antigen receptor (CAR)-based construct. The introduction of G72V mutation in the activation region of the TetR component of the rtTA further improved the fold response. Although the G72V mutation resulted in a removal of a cryptic splice site within rtTA, additional removal of this splice site led to only a modest improvement in the fold-response. Selective removal of key promoter elements (namely the BRE, TATA box, DPE and the four predicted Inr) confirmed the suitability of the minimal CMV promoter and its downstream sequences for supporting inducible expression. The results demonstrate marked improvement of the rtTA based Tet-On system in Sleeping Beauty for applications such as CAR T cell therapy.

Abnormal sarcolemmal Ca 2+ handling and impaired contractile function owing to sarcoplasmic reticulum/endoplasmic reticulum ATPase 2a (SERCA2a, encoded by ATP2A2 ) downregulation and/or dysfunction are pathognomonic hallmarks of heart failure (HF). There is, however, no clinically proven HF therapy that targets SERCA2a/ ATP2A2 dysregulation directly. Exploiting gene co-expression network analysis using an RNA sequencing dataset generated from human failing and non-failing left ventricular (LV) tissues, we identified long noncoding RNA lnc-SYNPO , transcribed from the 3’ untranslated region of SYNPO2 , as a potential regulator of SERCA2a/ ATP2A2 . Myocardial lnc-SYNPO was markedly upregulated during HF and its expression level showed a strong negative correlation (Pearson correlation coefficient=-0.60) with that of SERCA2a/ ATP2A2 in both mouse and human LV. Knockdown of lnc-SYNPO in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) increased, whereas overexpression of lnc-SYNPO reduced, SERCA2a/ ATP2A2 expression. In addition, lnc-SYNPO knockdown reversed endothelin-1 (ET-1) treatment-induced SERCA2a/ ATP2A2 downregulation, impaired Ca 2+ homeostasis and contractile dysfunction in hiPSC-CM. Mechanistically, the depletion of lnc-SYNPO, a chromatin-associated nuclear lncRNA, led to increased H3K4me3 and reduced H3K27me3 occupancy at SERCA2a/ ATP2A2 promoter. Chromatin isolation by RNA purification showed direct physical binding between lnc-SYNPO and the promoter of SERCA2a/ ATP2A2 . In addition, RNA pull-down assays showed that lnc-SYNPO binds to EZH2 and SUZ12, two core components of the polycomb repressor complex 2 (PRC2), suggesting that lnc-SYNPO represses SERCA2a/ ATP2A2 expression by recruiting PRC2 complex to the promoter of SERCA2a/ ATP2A2 , leading to epigenetic silencing of SERCA2a/ ATP2A2 . Taken together, these results unveiled a previously undiscovered epigenetic regulatory mechanism of SERCA2a/ ATP2A2 by lnc-SYNPO , contributing to impaired myocardial Ca 2+ homeostasis and contractile dysfunction observed with HF. Targeting lnc-SYNPO , therefore, could be a novel therapeutic approach to improve Ca 2+ homeostasis and contractile function in the failing human heart.

The present study investigates the genetic identification of Cinnamomum species by analyzing the nucleotide sequences of the partial non-coding internal transcribed spacer 2 (ITS2) of ribosomal genes. The genetic diversity of the five Cinnamomum species (Cinnamomum cassia, C. zeylanicum, C. loureiroi, C. osmophloeum and C. burmannii) was studied using polymerase chain reaction (PCR) amplification and a DNA sequencing method to molecularly identify the tested specimens. Phylogenetic trees were constructed using several clustering methods, including unweighted pair-group method with arithmetic average (UPGMA), neighbor-joining (NJ) and maximum evolution (ME). The eleven test samples were clustered into three major groups based on partial ITS2 sequences. The phylogenetic results showed that C. osmophloeum and C. burmanii are phylogenetically related to each other and that both are more closely related to C. cassia. Based on our results, there is enough sequence variation within the sequenced ITS2 regions to identify individual specimens among the five tested Cinnamomum species. Therefore, these ITS2 regions could be adopted as a molecular marker for differentiating among C. cassia, C. zeylanicum, C. loureiroi, C. osmophloem and C. burmannii.

OXA-48-like carbapenemase gene remains a hidden threat, as different OXA-48 variants have varying presentations of susceptibility to antibiotics that might affect the treatment decisions. Rapid detection and differentiation of OXA-48-like carbapenemase genes are critical for targeted treatment and infection control. In this study, we aimed to develop high-resolution melting (HRM) analysis for the differentiation of OXA-48 variants. HRM analysis is a post-polymerase chain reaction (post-PCR) method for identification of small variations in nucleic acid sequences based on the PCR dissociation curve. A total of 82 bacterial strains, which consisted of Enterobacteriaceae and non-Enterobacteriaceae, were collected from a tertiary teaching hospital. The sensitivity and specificity of the assay were determined, and the developed assay was evaluated using the collected isolates against conventional-sequencing method. Overall, the developed assay was able to detect isolates that harboured OXA-48 and OXA232/OXA-181 by showing two distinct peaks at 81.1 &plusmn, 0.2 &deg, C and 82.1 &plusmn, C, respectively. The detection limit of the assay was 1.6 x 10-6 ng/&micro, l for OXA-48 and 1.8 x 10-7 ng/&micro, l for OXA-232/OXA-181. This assay showed 100% specificity when evaluated on a panel of 37 isolates comprised of different species of bacteria and yeasts. When the assay with isolates collected in the year 2016 was first evaluated, the assay showed comparable results with conventional PCR-sequencing method where 34 OXA-48 and OXA-232/OXA-181 were detected. By using HRM analysis, the presence of OXA-48-like variants could be easily identified within 3 hours from the pure culture.

Mcl-1 is a member of the Bcl-2 anti-apoptotic protein family with important roles in the development, lifespan and metabolism of lymphocytes, as well as oncogenesis. Mcl-1 displays the shortest half-life of all Bcl-2 family members, with miRNA interference and proteasomal degradation being major pathways for Mcl-1 downregulation. In this study, we have identified a previously undescribed control mechanism active at the RNA level. A divergently transcribed lncRNA LOC107985203 (named here mcl1-AS1) negatively modulated Mcl-1 expression resulting in downregulation of Mcl-1 at both mRNA and protein level in a time-dependent manner. Using reporter assays, we confirmed that the mcl1-AS1 lncRNA promoter was located within Mcl-1 coding region. We next placed mcl1-AS1 under tetracycline-inducible control and demonstrated decreased viability in HEK293 cells upon doxycycline induction. Inhibition of mcl1-AS1 with shRNA reversed drug sensitivity. Bioinformatics surveys predicted direct mcl1-AS1 lncRNA binding to Mcl-1 transcripts, suggesting its mechanism in Mcl-1 expression is at the transcriptional level, consistent with a common role for anti-sense transcripts. The identification of a bi-directional promoter and lncRNA controlling Mcl-1 expression will have implications for controlling Mcl-1 activity in cancer cells, or for the purpose of enhancing the lifespan and quality of anti-cancer T lymphocytes.

Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for B cell malignancies, with emerging potential for the treatment of other hematologic cancers and solid tumors. The strength of the promoter within the CAR cassette will alter CAR-polypeptide levels on the cell surface of the T cell – impacting on the kinetics of activation, survival and memory cell formation in T cells. In addition to the CAR, promoters can be used to drive other genes of interest to enhance CAR T cell function. Expressing multiple genes from a single RNA transcript can be effectively achieved by linking the genes via a ribosomal skip site. However, promoters may differ in their ability to transcribe longer RNAs, or could interfere with lentiviral production, or transduction frequencies. In this study we compared the ability of the strong well-characterized promoters CMV, EF-1, hPGK and RPBSA to drive functional expression of a single RNA encoding three products: GFP, CAR, plus an additional cell-survival gene, Mcl-1. Although the four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of primary T cells. Major differences were found in the ability of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important consideration for gene therapy applications requiring the expression of long and complex mRNA.

Current models of mRNA turnover indicate that cytoplasmic degradation is coupled with translation. However, our understanding of the molecular events that coordinate ribosome transit with the mRNA decay machinery is still limited. Here, we show that the 4EHP–GIGYF1/2 complexes trigger co-translational mRNA decay as a result of perturbed elongation. Human cells lacking 4EHP and GIGYF1/2 proteins accumulate transcripts known to be degraded in a translation-dependent manner or with prominent ribosome pausing. These include among others, mRNAs encoding secretory and membrane-bound proteins or α- and β-tubulin subunits. In addition, 4EHP–GIGYF1/2 complexes fail to reduce target mRNA levels in the absence of ribosome stalling or upon disruption of their interaction with the mRNA cap structure, DDX6 or GYF domain-associated factors. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.

Alpinia coriandriodora is widely cultivated as a medicinal and edible Zingiberaceae plant in Guangxi, China. Since essential oils from Zingiberaceae plants can be used as a potential source of anticancer drugs, this study was designed to identify the chemical composition of A. coriandriodora rhizome essential oil (EO) and firstly elucidate its anticancer activities and related mechanisms. The GC-FID/MS results indicated that the most predominant component of EO was (E)-2-decenal (56.3%). Based on the MTT results, EO exhibited comparable cytotoxicity to A549 cells and lower cytotoxicity to non-cancerous cells compared with the positive control (elemene injection). For related mechanism studies, EO inhibited the A549 cells proliferation, resulting from inducing cell cycle arrest at G1 phase via downregulation of cyclin E2-CDK2 and cyclin D3-CDK4/CDK6 complexes and upregulation of p21. Meanwhile, it induced the apoptosis through mitochondrial pathway with the successive enhancement in the Bax/Bcl-2 ratio, Δψm decline, cyt c release, activation of caspase-9 and caspase-3, and PARP cleavage. Moreover, it inhibited the migration and invasion ability of A549 cells via downregulation of MMP-2 and N-cadherin, as well as upregulation of E-cadherin. Hence, EO has outstanding anticancer properties in vitro, and this fact encourages further studies using in vivo models.

Background Two recent genome-wide association studies (GWASs) reported that the FAM13A gene at the 4q22 locus associated with pulmonary fibrosis (defined by rs2609255) overlapping with COPD (defined by rs6837671). We hypothesized that single-nucleotide polymorphisms (SNPs) related to lung disease (especially pulmonary fibrosis) identified in this region are also associated with the risk of silicosis. Methods To test this hypothesis, we genotyped these two SNPs (rs2609255 and rs6837671) in a case-control study including 177 silicosis cases and 204 controls with silica dust exposure years similar to the levels for cases in a Chinese population. Results We found that rs2609255 was significantly associated with increased silicosis risk (dominant model: OR = 1.71; 95% CI = 1.01–2.92; P = 0.047). Additionally, eQTL analysis based on the GTEx database indicated that the rs2609255 polymorphism may alter the expression level of FAM13A in lung tissues (P = 1.8 × 10−4). Furthermore, interaction analyses showed that rs2609255 interacts multiplicatively with years of silica dust exposure to contribute to silicosis risk (interaction P = 0.040). Conclusions These results indicate that rs2609255 may modify silicosis susceptibility in the Chinese population.

Gaucher disease (GD) is a common lysosomal storage disorder caused by the deficiency of acid β-glucosidase, due to mutations in the GBA gene. To explore the clinical and molecular characteristics of GD patients from Southern China, GBA gene were analyzed by nest PCR and direct Sanger-sequencing. Novel missense mutations were transiently transfected in COS-7 cells by plasmid system for functional verification. Among the 22 GD patients, 19 patients were classified as type 1 and three as type 2. Over 60% of the type 1 patient had the onset before two years of age and about 42% of them died before three years of age. Six type 1 patients with L444P homozygous genotype, presented with early onset and severe hepatosplenomegaly. Four novel mutations Y22C, F109L, L149F and c.983_990delCCCACTGG were identified. The GBA activities in vitro of novel mutants Y22C, F109L and L149F were 20.2%, 6.9% and 6.5% of the wild-type, respectively. L444P mutation accounted for 47.7% of the mutant alleles. Our results revealed that type 1 GD tends to present with a severe phenotype among southern Chinese. L444P was the most prevalent mutation and L444P homozygous genotype was associated with severe type 1 GD. Three novel missense mutations identified were pathogenic.

Tendon repair is a clinical challenge because of the limited understanding on tenogenesis. The synthesis of type I collagen (Collagen I) and other extracellular matrix are essential for tendon differentiation and homeostasis. Current studies on tenogenesis focused mostly on the tenogenic transcriptional factors while the signaling controlling tenogenesis on translational level remains largely unknown. Here, we showed that mechanistic target of rapamycin (mTOR) signaling was activated by protenogenic growth factor, transforming growth factors beta1, and insulin-like growth factor-I. The expression of mTOR was upregulated during tenogenesis of mesenchymal stem cells (MSCs). Moreover, mTOR was downregulated in human tendinopathy tissues and was inactivated upon statin treatment. Both inhibition and depletion of AKT or mTOR significantly reduced type I collagen production and impaired tenogenesis of MSCs. Tendon specific-ablation of mTOR resulted in tendon defect and reduction of Collagen I. However, there is no evident downregulation of tendon associated collagens at the transcription level. Our study demonstrated that AKT-mTOR axis is a key mediator of tendon differentiation and provided a novel therapeutic target for tendinopathy and tendon injuries.

Summary: Fusion transcripts or RNAs have been found in both disordered and healthy human tissues and cells; however, their physiological functions in the brain development remain unknown. In the analysis of deposited RNA-sequence libraries covering early to middle embryonic stages, we identify 1,055 fusion transcripts present in the developing neocortex. Interestingly, 98 fusion transcripts exhibit distinct expression patterns in various neural progenitors (NPs) or neurons. We focus on CTNNBIP1-CLSTN1 (CTCL), which is enriched in outer radial glial cells that contribute to cortex expansion during human evolution. Intriguingly, downregulation of CTCL in cultured human cerebral organoids causes marked reduction in NPs and precocious neuronal differentiation, leading to impairment of organoid growth. Furthermore, the expression of CTCL fine-tunes Wnt/β-catenin signaling that controls cortex patterning. Together, this work provides evidence indicating important roles of fusion transcript in human brain development and evolution.

Three new dolabranes, tagalenes G − I (1 − 3), together with five known analogues (4 − 8), were isolated from the twigs of the Chinese mangrove, Ceriops tagal. Their structures were determined on the basis of extensive spectroscopic analysis and the comparison with the previous data. Tagalene G (1) is a 16-nordolabrane with a 15-hydroxymethyl scaffold, tagalene H (2) possesses an 18-hydroxymethyl moiety, whereas tagalene I (3) is a further addition to the small group of dolabranes with a rare 4, 18-epoxy ring. Compounds 6 − 7 were isolated from C. tagal for the first time.

Sulfur dioxide (SO2) is a common air pollutant that has adverse effects on plants. MicroRNAs (miRNAs) are small noncoding RNA that play critical roles in plant development and stress response. In this study, we found that two miRNAs, miR398 and miR395, were differentially expressed in Arabidopsis shoots under SO2 stress. The expression of miR398 was down-regulated, and the transcript levels of its target genes, Cu/Zn superoxide dismutases (CSD1 and CSD2), were increased during SO2 exposure. The activity of superoxide dismutase (SOD), one of the major antioxidant enzymes, was enhanced with the increase in the CSD transcript level, suggesting an important role of miR398 in response to SO2-induced oxidative stress. Meanwhile, the expression of miR395 was increased, and the transcript levels of its target genes, ATP sulfurylases (APS3 and APS4) and a low-affinity sulfate transporter (SULTR2;1), were decreased in Arabidopsis shoots, showing that miR395 played important roles in the regulation of sulfate assimilation and translocation during SO2 exposure. The content of glutathione (GSH), an important sulfur-containing antioxidant, was enhanced with the changes in sulfur metabolism in Arabidopsis shoots under SO2 stress. These results showed that both miR398 and miR395 were involved in protecting plants from oxidative damage during SO2 exposure. Many stress-responsive cis-elements were found in the promoter regions of MIR398 and MIR395, suggesting that these miRNAs might respond to various environmental conditions, including SO2 stress. Overall, our study provides an insight into the regulatory roles of miRNAs in response to SO2 stress in plants, and highlights the molecular mechanisms of plant adaptation to environmental stress.

Purpose: Low molecular weight cyclin E (LMW-E) detected by Western blot analysis predicts for reduced breast cancer survival; however, it is impractical for clinical use. LMW-E lacks a nuclear localization signal that leads to accumulation in the cytoplasm that can be detected by IHC. We tested the hypothesis that cytoplasmic staining of cyclin E can be used as a predictor of poor outcome in different subtypes of breast cancer using patient cohorts with distinct clinical and pathologic features.Experimental Design: We evaluated the subcellular localization of cyclin E in breast cancer specimens from 2,494 patients from 4 different cohorts: 303 from a prospective study and 2,191 from retrospective cohorts [NCI, MD Anderson Cancer Center (MDA), and the United Kingdom (UK)]. Median follow-up times were 8.0, 10.1, 13.5, and 5.7 years, respectively.Results: Subcellular localization of cyclin E on IHC was associated with full-length (nuclear) and low molecular weight isoforms (cytoplasmic) of cyclin E on Western blot analysis. In multivariable analysis, cytoplasmic cyclin E staining was associated with the greatest risk of recurrence compared with other prognostic factors across all subtypes in three (NCI, MDA, and UK) of the cohorts. In the MDA cohort, cytoplasmic cyclin E staining outperformed Ki67 and all other variables as prognostic factors.Conclusions: Cytoplasmic cyclin E identifies patients with the highest likelihood of recurrence consistently across different patient cohorts and subtypes. These patients may benefit from alternative therapies targeting the oncogenic isoforms of cyclin E. Clin Cancer Res; 23(12); 2991–3002. ©2016 AACR.

Extradomain-B fibronectin (EDB-FN), an oncofetal isoform of FN, is a promising diagnostic and therapeutic target of tumors, including breast cancer. Many EDB-FN-targeted drugs have been developed and have shown therapeutic effects in clinical trials. Molecular imaging to visualize EDB-FN-positive cancers may help select the right patients who will be benefit from EDB-FN-targeted therapy. Although a few EDB-FN-targeted imaging probes have been developed, the complicated manufacturing procedure and expensive material and equipment required limit their application for large-scale screening of EDB-FN-positive cancer patients. Thus, more simple and economic EDB-FN-targeted imaging probes are still urgently needed. Previously, we have identified a breast cancer-targeted peptide, CTVRTSADC. Coincidently, it was later identified as an EDB-FN-targeted peptide and named ZD2. In this study, we found a positive correlation between the binding activity of the ZD2 phage and the expression level of EDB-FN in breast canc...