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Fulltext Verifying Death Reports in the Platelet Inhibition and Patient Outcomes (PLATO) Trial

Serebruany V, Tanguay JF, Benavides MA, Cabrera-Fuentes H, Eisert W, Kim MH, et al.

Am J Ther, 2020 10 29;27(6):e563-e572.
PMID: 33109913 DOI: 10.1097/MJT.0000000000001286

BACKGROUND: Excess vascular deaths in the PLATO trial comparing ticagrelor to clopidogrel have been repeatedly challenged by the Food and Drug Administration (FDA) reviewers and academia. Based on the Freedom of Information Act, BuzzFeed won a court order and shared with us the complete list of reported deaths for the ticagrelor FDA New Drug Application (NDA) 22-433. This dataset was matched against local patient-level records from PLATO sites monitored by the sponsor.
STUDY QUESTION: Whether FDA death data in the PLATO trial matched the local site records.
STUDY DESIGN: The NDA spreadsheet contains 938 precisely detailed PLATO deaths. We obtained and validated local evidence for 52 deaths among 861 PLATO patients from 14 enrolling sites in 8 countries and matched those with the official NDA dataset submitted to the FDA.
MEASURES AND OUTCOMES: Existence, precise time, and primary cause of deaths in PLATO.
RESULTS: Discrepant to the NDA document, sites confirmed 2 extra unreported deaths (Poland and Korea) and failed to confirm 4 deaths (Malaysia). Of the remaining 46 deaths, dates were reported correctly for 42 patients, earlier (2 clopidogrel), or later (2 ticagrelor) than the actual occurrence of death. In 12 clopidogrel patients, cause of death was changed to "vascular," whereas 6 NDA ticagrelor "nonvascular" or "unknown" deaths were site-reported as of "vascular" origin. Sudden death was incorrectly reported in 4 clopidogrel patients, but omitted in 4 ticagrelor patients directly affecting the primary efficacy PLATO endpoint.
CONCLUSIONS: Many deaths were inaccurately reported in PLATO favoring ticagrelor. The full extent of mortality misreporting is currently unclear, while especially worrisome is a mismatch in identifying primary death cause. Because all PLATO events are kept in the cloud electronic Medidata Rave capture system, securing the database content, examining the dataset changes or/and repeated entries, identifying potential interference origin, and assessing full magnitude of the problem are warranted.
Tunable metasurfaces for visible and SWIR applications

Lee CW, Choi HJ, Jeong H

Nano Converg, 2020 Jan 20;7(1):3.
PMID: 31956942 DOI: 10.1186/s40580-019-0213-2

Demand on optical or photonic applications in the visible or short-wavelength infrared (SWIR) spectra, such as vision, virtual or augmented displays, imaging, spectroscopy, remote sensing (LIDAR), chemical reaction sensing, microscopy, and photonic integrated circuits, has envisaged new type of subwavelength-featured materials and devices for controlling electromagnetic waves. The study on metasurfaces, of which the thickness is either comparable to or smaller than the wavelength of the considered incoming electromagnetic wave, has been grown rapidly to embrace the needs of developing sub 100-micron active photonic pixelated devices and their arrayed form. Meta-atoms in metasurfaces are now actively controlled under external stimuli to lead to a large phase shift upon the incident light, which has provided a huge potential for arrayed two-dimensional active optics. This short review summarizes actively tunable or reconfigurable metasurfaces for the visible or SWIR spectra, to account for the physical operating principles and the current issues to overcome.
Fulltext Tumor Necrosis Factor-Alpha Exacerbates Viral Entry in SARS-CoV2-Infected iPSC-Derived Cardiomyocytes

Lee CY, Huang CH, Rastegari E, Rengganaten V, Liu PC, Tsai PH, et al.

Int J Mol Sci, 2021 Sep 13;22(18).
PMID: 34576032 DOI: 10.3390/ijms22189869

The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.
Fulltext Trial of everolimus-eluting stents or bypass surgery for coronary disease

Park SJ, Ahn JM, Kim YH, Park DW, Yun SC, Lee JY, et al.

N Engl J Med, 2015 Mar 26;372(13):1204-12.
PMID: 25774645 DOI: 10.1056/NEJMoa1415447

BACKGROUND: Most trials comparing percutaneous coronary intervention (PCI) with coronary-artery bypass grafting (CABG) have not made use of second-generation drug-eluting stents.
METHODS: We conducted a randomized noninferiority trial at 27 centers in East Asia. We planned to randomly assign 1776 patients with multivessel coronary artery disease to PCI with everolimus-eluting stents or to CABG. The primary end point was a composite of death, myocardial infarction, or target-vessel revascularization at 2 years after randomization. Event rates during longer-term follow-up were also compared between groups.
RESULTS: After the enrollment of 880 patients (438 patients randomly assigned to the PCI group and 442 randomly assigned to the CABG group), the study was terminated early owing to slow enrollment. At 2 years, the primary end point had occurred in 11.0% of the patients in the PCI group and in 7.9% of those in the CABG group (absolute risk difference, 3.1 percentage points; 95% confidence interval [CI], -0.8 to 6.9; P=0.32 for noninferiority). At longer-term follow-up (median, 4.6 years), the primary end point had occurred in 15.3% of the patients in the PCI group and in 10.6% of those in the CABG group (hazard ratio, 1.47; 95% CI, 1.01 to 2.13; P=0.04). No significant differences were seen between the two groups in the occurrence of a composite safety end point of death, myocardial infarction, or stroke. However, the rates of any repeat revascularization and spontaneous myocardial infarction were significantly higher after PCI than after CABG.
CONCLUSIONS: Among patients with multivessel coronary artery disease, the rate of major adverse cardiovascular events was higher among those who had undergone PCI with the use of everolimus-eluting stents than among those who had undergone CABG. (Funded by CardioVascular Research Foundation and others; BEST ClinicalTrials.gov number, NCT00997828.).
Fulltext The uS8, uS4, eS31, and uL14 Ribosomal Protein Genes Are Dysregulated in Nasopharyngeal Carcinoma Cell Lines

Sim EU, Ng KL, Lee CW, Narayanan K

Biomed Res Int, 2017;2017:4876954.
PMID: 28791303 DOI: 10.1155/2017/4876954

The association of ribosomal proteins with carcinogenesis of nasopharyngeal carcinoma (NPC) has been established in a limited subset of ribosomal protein genes. To date, three ribosomal protein genes, eL27 (L27), eL41 (L41), and eL43 (L37a), have been found to be differentially expressed in cell lines derived from NPC tumors. This raises the possibility of more ribosomal protein genes that could be associated with NPC. In this study, we investigated the expression profiles of eight ribosomal protein genes, uS8 (S8), uS4 (S9), eS31 (S27a), eL6 (L6), eL18 (L18), uL14 (L23), eL24 (L24), and eL30 (L30), in six NPC-derived cell lines (HONE-1, SUNE1, HK1, TW01, TW04, and C666-1). Their expression levels were compared with that of a nonmalignant nasopharyngeal epithelial cell line (NP69) using quantitative real-time PCR (RT-qPCR) assay. Of the eight genes studied, the expressions of four ribosomal protein genes uS8 (S8), uS4 (S9), eS31 (S27a), and uL14 (L23) were found to be significantly downregulated in NPC cell lines relative to NP69. Our findings provide novel empirical evidence of these four ribosomal protein genes as NPC-associated genetic factors and reinforce the relevance of ribosomal proteins in the carcinogenesis of nasopharyngeal cancer.
Fulltext The roles of ribosomal proteins in nasopharyngeal cancer: culprits, sentinels or both

Sim EU, Lee CW, Narayanan K

Biomark Res, 2021 Jun 30;9(1):51.
PMID: 34193301 DOI: 10.1186/s40364-021-00311-x

Ribosomal protein genes encode products that are essential for cellular protein biosynthesis and are major components of ribosomes. Canonically, they are involved in the complex system of ribosome biogenesis pivotal to the catalysis of protein translation. Amid this tightly organised process, some ribosomal proteins have unique spatial and temporal physiological activity giving rise to their extra-ribosomal functions. Many of these extra-ribosomal roles pertain to cellular growth and differentiation, thus implicating the involvement of some ribosomal proteins in organogenesis. Consequently, dysregulated functions of these ribosomal proteins could be linked to oncogenesis or neoplastic transformation of human cells. Their suspected roles in carcinogenesis have been reported but not specifically explained for malignancy of the nasopharynx. This is despite the fact that literature since one and half decade ago have documented the association of ribosomal proteins to nasopharyngeal cancer. In this review, we explain the association and contribution of dysregulated expression among a subset of ribosomal proteins to nasopharyngeal oncogenesis. The relationship of these ribosomal proteins with the cancer are explained. We provide information to indicate that the dysfunctional extra-ribosomal activities of specific ribosomal proteins are tightly involved with the molecular pathogenesis of nasopharyngeal cancer albeit mechanisms yet to be precisely defined. The complete knowledge of this will impact future applications in the effective management of nasopharyngeal cancer.
The impact of eutrophication towards selected bacterial process rates in tropical coastal waters

Lim JH, Lee CW, Bong CW, Kudo I

Mar Pollut Bull, 2021 May 25;169:112524.
PMID: 34049069 DOI: 10.1016/j.marpolbul.2021.112524

The dissolved organic nutrient conditions and bacterial process rates at two tropical coastal sites in Peninsular Malaysia (Port Klang and Port Dickson) were initially studied in 2004-2005 period and later revisited in 2010-2011. We observed that dissolved organic nitrogen (DON) increased about two- and ten-fold at Port Klang and Port Dickson, respectively and resulted in a significant change in DOC:DON ratio (t ≥ 2.077, p
Fulltext The Monkey Head Mushroom and Memory Enhancement in Alzheimer's Disease

Yanshree, Yu WS, Fung ML, Lee CW, Lim LW, Wong KH

Cells, 2022 Jul 24;11(15).
PMID: 35892581 DOI: 10.3390/cells11152284

Alzheimer's disease (AD) is a neurodegenerative disorder, and no effective treatments are available to treat this disorder. Therefore, researchers have been investigating Hericium erinaceus, or the monkey head mushroom, an edible medicinal mushroom, as a possible treatment for AD. In this narrative review, we evaluated six preclinical and three clinical studies of the therapeutic effects of Hericium erinaceus on AD. Preclinical trials have successfully demonstrated that extracts and bioactive compounds of Hericium erinaceus have potential beneficial effects in ameliorating cognitive functioning and behavioral deficits in animal models of AD. A limited number of clinical studies have been conducted and several clinical trials are ongoing, which have thus far shown analogous outcomes to the preclinical studies. Nonetheless, future research on Hericium erinaceus needs to focus on elucidating the specific neuroprotective mechanisms and the target sites in AD. Additionally, standardized treatment parameters and universal regulatory systems need to be established to further ensure treatment safety and efficacy. In conclusion, Hericium erinaceus has therapeutic potential and may facilitate memory enhancement in patients with AD.
Temporal variation of phytoplankton growth and grazing loss in the west coast of Peninsular Malaysia

Lim JH, Lee CW, Kudo I

Environ Monit Assess, 2015 May;187(5):246.
PMID: 25864082 DOI: 10.1007/s10661-015-4487-5

Phytoplankton growth (μ) and grazing loss (g) rates were measured monthly by the Landry-Hassett dilution method over a 2-year period at both estuarine (Klang) and coastal water (Port Dickson) systems along the Straits of Malacca. Chlorophyll a (Chl a) concentration ranged from 0.20 to 4.47 μg L(-1) at Klang except on two occasions when Chl a spiked above 10 μg L(-1). In contrast, Chl a concentrations were relatively stable at Port Dickson (0.14 to 2.76 μg L(-1)). From the rate measurements, μ was higher (t = 2.01, df = 43, p 0.80). g ranged from 0.30 to 1.50 and 0.21 to 1.51 day(-1) at Klang and Port Dickson, respectively. In this study, grazing loss was coupled to phytoplankton growth, and the ratio of g/μ or grazing pressure which estimates the proportion of primary production grazed was 50% at Klang and lower than at Port Dickson (68%; t = 2.213, df = 36, p
Fulltext Temporal variation of bacterial respiration and growth efficiency in tropical coastal waters

Lee CW, Bong CW, Hii YS

Appl Environ Microbiol, 2009 Dec;75(24):7594-601.
PMID: 19820145 DOI: 10.1128/AEM.01227-09

We investigated the temporal variation of bacterial production, respiration, and growth efficiency in the tropical coastal waters of Peninsular Malaysia. We selected five stations including two estuaries and three coastal water stations. The temperature was relatively stable (averaging around 29.5 degrees C), whereas salinity was more variable in the estuaries. We also measured dissolved organic carbon and nitrogen (DOC and DON, respectively) concentrations. DOC generally ranged from 100 to 900 microM, whereas DON ranged from 0 to 32 microM. Bacterial respiration ranged from 0.5 to 3.2 microM O2 h(-1), whereas bacterial production ranged from 0.05 to 0.51 microM C h(-1). Bacterial growth efficiency was calculated as bacterial production/(bacterial production + respiration), and ranged from 0.02 to 0.40. Multiple correlation analyses revealed that bacterial production was dependent upon primary production (r2 = 0.169, df = 31, and P < 0.02) whereas bacterial respiration was dependent upon both substrate quality (i.e., DOC/DON ratio) (r2 = 0.137, df = 32, and P = 0.03) and temperature (r2 = 0.113, df = 36, and P = 0.04). Substrate quality was the most important factor (r2 = 0.119, df = 33, and P = 0.04) for the regulation of bacterial growth efficiency. Using bacterial growth efficiency values, the average bacterial carbon demand calculated was from 5.30 to 11.28 microM C h(-1). When the bacterial carbon demand was compared with primary productivity, we found that net heterotrophy was established at only two stations. The ratio of bacterial carbon demand to net primary production correlated significantly with bacterial growth efficiency (r2 = 0.341, df = 35, and P < 0.001). From nonlinear regression analysis, we found that net heterotrophy was established when bacterial growth efficiency was <0.08. Our study showed the extent of net heterotrophy in these waters and illustrated the importance of heterotrophic microbial processes in coastal aquatic food webs.
Synechococcus production and grazing loss rates in nearshore tropical waters

Heng PL, Lim JH, Lee CW

Environ Monit Assess, 2017 Mar;189(3):117.
PMID: 28220442 DOI: 10.1007/s10661-017-5838-1

Temporal variation of Synechococcus, its production (μ) and grazing loss (g) rates were studied for 2 years at nearshore stations, i.e. Port Dickson and Port Klang along the Straits of Malacca. Synechococcus abundance at Port Dickson (0.3-2.3 × 10(5) cell ml(-1)) was always higher than at Port Klang (0.3-7.1 × 10(4) cell ml(-1)) (p 0.25), but nutrient and light availability were important factors for their distribution. The relationship was modelled as log Synechococcus = 0.37Secchi - 0.01DIN + 4.52 where light availability (as Secchi disc depth) was a more important determinant. From a two-factorial experiment, nutrients were not significant for Synechococcus growth as in situ nutrient concentrations exceeded the threshold for saturated growth. However, light availability was important and elevated Synechococcus growth rates especially at Port Dickson (F = 5.94, p 0.30). In nearshore tropical waters, an estimated 69 % of Synechococcus production could be grazed.
Storage and dynamics of soil organic carbon in allochthonous-dominated and nitrogen-limited natural and planted mangrove forests in southern Thailand

Hu J, Pradit S, Loh PS, Chen Z, Guo C, Le TPQ, et al.

Mar Pollut Bull, 2024 Jan 29;200:116064.
PMID: 38290368 DOI: 10.1016/j.marpolbul.2024.116064

Mangrove forests can help to mitigate climate change by storing a significant amount of carbon (C) in soils. Planted mangrove forests have been established to combat anthropogenic threats posed by climate change. However, the efficiency of planted forests in terms of soil organic carbon (SOC) storage and dynamics relative to that of natural forests is unclear. We assessed SOC and nutrient storage, SOC sources and drivers in a natural and a planted forest in southern Thailand. Although the planted forest stored more C and nutrients than the natural forest, the early-stage planted forest was not a strong sink relative to mudflat. Both forests were predominated by allochthonous organic C and nitrogen limited, with total nitrogen being a major driver of SOC in both cases. SOC showed a significant decline along land-to-sea and depth gradients as a result of soil texture, nutrient availability, and pH in the natural forest.
Fulltext Sphingosine 1-phosphate receptor 2 (S1P2) attenuates reactive oxygen species formation and inhibits cell death: implications for otoprotective therapy

Herr DR, Reolo MJ, Peh YX, Wang W, Lee CW, Rivera R, et al.

Sci Rep, 2016 Apr 15;6:24541.
PMID: 27080739 DOI: 10.1038/srep24541

Ototoxic drugs, such as platinum-based chemotherapeutics, often lead to permanent hearing loss through apoptosis of neuroepithelial hair cells and afferent neurons of the cochlea. There is no approved therapy for preventing or reversing this process. Our previous studies identified a G protein-coupled receptor (GPCR), S1P2, as a potential mediator of otoprotection. We therefore sought to identify a pharmacological approach to prevent cochlear degeneration via activation of S1P2. The cochleae of S1pr2(-/-) knockout mice were evaluated for accumulation of reactive oxygen species (ROS) with a nitro blue tetrazolium (NBT) assay. This showed that loss of S1P2 results in accumulation of ROS that precedes progressive cochlear degeneration as previously reported. These findings were supported by in vitro cell-based assays to evaluate cell viability, induction of apoptosis, and accumulation of ROS following activation of S1P2 in the presence of cisplatin. We show for the first time, that activation of S1P2 with a selective receptor agonist increases cell viability and reduces cisplatin-mediated cell death by reducing ROS. Cumulatively, these results suggest that S1P2 may serve as a therapeutic target for attenuating cisplatin-mediated ototoxicity.
Short homologies efficiently generate detectable homologous recombination events

Osahor AN, Tan CY, Sim EU, Lee CW, Narayanan K

Anal Biochem, 2014 Oct 1;462:26-8.
PMID: 24929088 DOI: 10.1016/j.ab.2014.05.030

When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.
Fulltext Sarcopenia is a useful risk stratification tool to prognosticate splenic abscess patients in the emergency department

Hung SK, Kou HW, Hsu KH, Wu CT, Lee CW, Leonard Goh ZN, et al.

J Formos Med Assoc, 2021 Mar;120(3):997-1004.
PMID: 32917483 DOI: 10.1016/j.jfma.2020.08.039

BACKGROUND/PURPOSE: Splenic abscess is a life-threatening surgical emergency which requires early diagnosis and intervention to maximize patient outcomes. This can be achieved through accurate risk stratification in the emergency department (ED). Sarcopenia refers to an age-related loss of skeletal muscle mass and strength that is accompanied by major physiologic and clinical ramifications, and often signifies decreased physiologic reserves. It is associated with poor clinical outcomes in sepsis, acute respiratory failure, oncological surgery, and liver transplantation. This study evaluates the utility of sarcopenia as a radiological stratification tool to predict in-hospital mortality of splenic abscess patients in the ED. This will assist emergency physicians, internists and surgeons in rapid risk stratification, assessing treatment options, and communicating with family members.
METHODS: 99 adult patients at four training and research hospitals who had undergone an abdominal contrast computed tomography scan in the ED with the final diagnosis of splenic abscess from January 2004 to November 2017 were recruited. Evaluation for sarcopenia was performed via calculating the psoas cross-sectional area at the level of the third lumbar vertebra and normalising for height, before checking it against pre-defined values. Univariate analyses were used to evaluate the differences between survivors and non-survivors. Sensitivity, specificity, and predictive values of the presence of sarcopenia in predicting in-hospital mortality were calculated. Kaplan-Meier methods, log-rank test, and Cox proportional hazards model were also performed to examine survival between groups with sarcopenia versus non-sarcopenia.
RESULTS: Splenic abscess patients with sarcopenia were 7.56 times more at risk of in-hospital mortality than those without sarcopenia (multivariate-adjusted HR: 7.56; 95% CI: 1.55-36.93). Presence of sarcopenia was found to have 84.62% sensitivity and 96.49% negative predictive value in predicting mortality.
CONCLUSION: Sarcopenia is associated with poor prognoses of in-hospital mortality in patients with splenic abscess presenting to the ED. We recommend its use in the ED to rapidly risk stratify and predict outcome to guide treatment strategies.
Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis

Osahor A, Deekonda K, Lee CW, Sim EU, Radu A, Narayanan K

Anal Biochem, 2017 10 01;534:46-48.
PMID: 28693990 DOI: 10.1016/j.ab.2017.07.008

Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.
RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells

Akinsola RO, Adewoyin M, Lee CW, Sim EU, Narayanan K

Anal Biochem, 2021 12 01;634:114432.
PMID: 34695391 DOI: 10.1016/j.ab.2021.114432

Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based high-throughput and inexpensive method for tracking bacterial adherence and internalization through flow cytometry to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and cell-dependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study host-bacteria interaction.
Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells

Wong YC, Ng AWR, Chen Q, Liew PS, Lee CW, Sim EUH, et al.

ACS Synth Biol, 2023 Apr 21;12(4):909-921.
PMID: 37026178 DOI: 10.1021/acssynbio.2c00580

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.
Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells

Liew PS, Chen Q, Ng AWR, Chew YC, Ravin NV, Sim EUH, et al.

Anal Biochem, 2019 10 15;583:113361.
PMID: 31306622 DOI: 10.1016/j.ab.2019.113361

Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.
Fulltext Monitoring Consumption of Common Illicit Drugs in Kuala Lumpur, Malaysia, by Wastewater-Cased Epidemiology

Du P, Liu X, Zhong G, Zhou Z, Thomes MW, Lee CW, et al.

Int J Environ Res Public Health, 2020 01 31;17(3).
PMID: 32023897 DOI: 10.3390/ijerph17030889

Southeast Asian countries including Malaysia play a major role in global drug trade and abuse. Use of amphetamine-type stimulants has increased in the past decade in Malaysia. This study aimed to apply wastewater-based epidemiology for the first time in Kuala Lumpur, Malaysia, to estimate the consumption of common illicit drugs in urban population. Influent wastewater samples were collected from two wastewater treatment plants in Kuala Lumpur in the summer of 2017. Concentrations of twenty-four drug biomarkers were analyzed for estimating drug consumption. Fourteen drug residues were detected with concentrations of up to 1640 ng/L. Among the monitored illicit drugs, 3,4-methylenedioxy-methamphetamine (MDMA) or ecstasy had the highest estimated per capita consumptions. Consumption and dose of amphetamine-type stimulants (methamphetamine and MDMA) were both an order of magnitude higher than those of opioids (heroin and codeine, methadone and tramadol). Amphetamine-type stimulants were the most prevalent drugs, replacing opioids in the drug market. The prevalence trend measured by wastewater-based epidemiology data reflected the shift to amphetamine-type stimulants as reported by the Association of Southeast Asian Nations Narcotics Cooperation Center. Most of the undetected drug residues were new psychoactive substances (NPSs), suggesting a low prevalence of NPSs in the drug market.

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