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Neurobiology of Kratom and its main alkaloid mitragynine

Suhaimi FW, Yusoff NH, Hassan R, Mansor SM, Navaratnam V, Müller CP, et al.

Brain Res Bull, 2016 09;126(Pt 1):29-40.
PMID: 27018165 DOI: 10.1016/j.brainresbull.2016.03.015

Kratom or its main alkaloid, mitragynine is derived from the plant Mitragyna speciosa Korth which is indigenous to Southeast Asian countries. This substance has become widely available in other countries like Europe and United States due to its opium- and coca-like effects. In this article, we have reviewed available reports on mitragynine and other M. speciosa extracts. M. speciosa has been proven to have a rewarding effect and is effective in alleviating the morphine and ethanol withdrawal effects. However, studies in human revealed that prolonged consumption of this plant led to dependence and tolerance while cessation caused a series of aversive withdrawal symptoms. Findings also showed that M. speciosa extracts possess antinociceptive, anti-inflammatory, anti-depressant, and muscle relaxant properties. Available evidence further supports the adverse effects of M. speciosa preparations, mitragynine on cognition. Pharmacological activities are mainly mediated via opioid receptors as well as neuronal Ca2+ channels, expression of cAMP and CREB protein and via descending monoaminergic system. Physicochemical properties of mitragynine have been documented which may further explain the variation in pharmacological responses. In summary, current researchs on its main indole alkaloid, mitragynine suggest both therapeutic and addictive potential but further research on its molecular effects is needed.
Fulltext Frontal Electroencephalogram Alpha Asymmetry during Mental Stress Related to Workplace Noise

Alyan E, Saad NM, Kamel N, Yusoff MZ, Zakariya MA, Rahman MA, et al.

Sensors (Basel), 2021 Mar 11;21(6).
PMID: 33799722 DOI: 10.3390/s21061968

This study aims to investigate the effects of workplace noise on neural activity and alpha asymmetries of the prefrontal cortex (PFC) during mental stress conditions. Workplace noise exposure is a pervasive environmental pollutant and is negatively linked to cognitive effects and selective attention. Generally, the stress theory is assumed to underlie the impact of noise on health. Evidence for the impacts of workplace noise on mental stress is lacking. Fifteen healthy volunteer subjects performed the Montreal imaging stress task in quiet and noisy workplaces while their brain activity was recorded using electroencephalography. The salivary alpha-amylase (sAA) was measured before and immediately after each tested workplace to evaluate the stress level. The results showed a decrease in alpha rhythms, or an increase in cortical activity, of the PFC for all participants at the noisy workplace. Further analysis of alpha asymmetry revealed a greater significant relative right frontal activation of the noisy workplace group at electrode pairs F4-F3 but not F8-F7. Furthermore, a significant increase in sAA activity was observed in all participants at the noisy workplace, demonstrating the presence of stress. The findings provide critical information on the effects of workplace noise-related stress that might be neglected during mental stress evaluations.
Fulltext Unilateral neglect or alien hand syndrome? A diagnostic challenge

Wan Yusoff WR, Hanafi MH, Ibrahim AH, Kassim NK, Suhaimi A

J Taibah Univ Med Sci, 2021 Apr;16(2):288-291.
PMID: 33897336 DOI: 10.1016/j.jtumed.2020.12.003

Alien hand syndrome (AHS) is a rare post-stroke complication. Although the occurrence of AHS after stroke is rare, it can have a disabling impact on the lifestyle and career of the patients post stroke. It is difficult to distinguish AHS from the non-dominant hemispheric infarction with symptoms of hemianopia and left hemineglect, as patients with AHS can also have neglect as well as behaviour symptoms if the frontal lobe is involved. We report the case of a 62-year-old gentleman who was admitted to the rehabilitation ward and was treated for neglect and apraxia following right middle cerebral artery stroke. After further re-evaluation, the patient was diagnosed with AHS. Functionally, he improved and the symptoms disappeared four months after the stroke. In this case report, we highlight the features of AHS and provide a unique management approach.
Optimal implementation of low impact development for urban stormwater quantity and quality control using multi-objective optimization

Rezaei AR, Ismail Z, Niksokhan MH, Dayarian MA, Ramli AH, Yusoff S

Environ Monit Assess, 2021 Mar 31;193(4):241.
PMID: 33791871 DOI: 10.1007/s10661-021-09010-4

Stormwater runoff is a major concern in urban areas which is mostly the result of vast urbanization. To reduce urban stormwater runoff and improve water quality, low impact development (LID) is used in urban areas. Therefore, it is vital to find the optimal combination of LID controls to achieve maximum reduction in both stormwater runoff and pollutants with optimal cost. In this study, a simulation-optimization model was developed by linking the EPA Storm Water Management Model (SWMM) to the Multi-Objective Particle Swarm Optimization (MOPSO) using MATLAB. The coupled model could carry out multi-objective optimization (MOO) and find potential solutions to the optimization objectives using the SWMM simulation model outputs. The SWMM model was developed using data from the BUNUS catchment in Kuala Lumpur, Malaysia. The total suspended solids (TSS) and total nitrogen (TN) were selected as pollutants to be used in the simulation model. Vegetated swale and rain garden were selected as LID controls for the study area. The LID controls were assigned to the model using the catchment characteristics. The target objectives were to minimize peak stormwater runoff, TSS, and TN with the minimum number of LID controls applications. The LID combination scenarios were also tested in SWMM to identify the best LID types and combination to achieve maximum reduction in both peak runoff and pollutants. This study found that the peak runoff, TSS, and TN were reduced by 13%, 38%, and 24%, respectively. The optimal number of LID controls that could be used at the BUNUS catchment area was also found to be 25.
Fulltext Crystal structure of 4-({(1E,2E)-3-[3-(4-fluoro-phen-yl)-1-isopropyl-1H-indol-2-yl]allyl-idene}amino)-5-methyl-1H-1,2,4-triazole-5(4H)-thione

Kulkarni AD, Rahman ML, Mohd Yusoff M, Kwong HC, Quah CK

Acta Crystallogr E Crystallogr Commun, 2015 Nov 1;71(Pt 11):1411-3.
PMID: 26594522 DOI: 10.1107/S2056989015020101

The title compound, C23H22FN5S, exists in a trans conformation with respect to the methene C=C and the acyclic N=C bonds. The 1,2,4-triazole-5(4H)-thione ring makes dihedral angles of 88.66 (9) and 84.51 (10)°, respectively, with the indole and benzene rings. In the crystal, mol-ecules are linked by pairs of N-H⋯S hydrogen bonds, forming inversion dimers with an R 2 (2)(8) ring motif. The dimers are linked via C-H⋯π inter-actions, forming chains along [1-10]. The chains are linked via π-π inter-actions involving inversion-related triazole rings [centroid-centroid distance = 3.4340 (13) Å], forming layers parallel to the ab plane.
Improving the efficiency of post-digestion method in extracting microplastics from gastrointestinal tract and gills of fish

Jaafar N, Musa SM, Azfaralariff A, Mohamed M, Yusoff AH, Lazim AM

Chemosphere, 2020 Dec;260:127649.
PMID: 32688323 DOI: 10.1016/j.chemosphere.2020.127649

Post-digestion treatment is an important step during sample preparation to facilitate the removal of undigested materials for better detection of ingested microplastics. Sieving, density separation with zinc chloride solution (ZnCl2), and oil extraction protocol (OEP) have been introduced in separating microplastics from sediments. The clean-up methods are rarely highlighted in previous studies, especially in the separation of microplastics from marine biota. Thus, this study proposed and compared the suitability of three techniques, which can reduce the number of undigested particles from the digestate of GIT and gills. Our result has shown excellent removal of non-plastics materials and reduces the coloration of filter paper in all treated samples. Both sieving and density separation achieved optimum post-digestion efficiencies of >95% for both GIT and gill samples, which former showed no effect on polymer integrity. Additionally, high recovery rate was obtained for the larger size microplastics (>500 μm) with approximately 97.7% (GIT) and 95.7% (gill), respectively. Exposure to the ZnCl2 solution led to a significant loss of smaller size PET and changed the absorption spectrums of all tested polymers. Particle morphology determined by SEM revealed such exposure eroded the surface of PET fragments and elemental analysis has shown detectable peaks of zinc and chlorine appeared. Low microplastics recoveries were achieved through OPE and residue of oil was observed from the infrared spectrum of all tested polymer. The findings demonstrate sieving with size fractioning can provide exceptional removal of non-plastics materials from the digestate of GIT and gill samples.
Mucosal responses of brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) following intraperitoneal infection with Vibrio harveyi

Chieng CCY, Daud HM, Yusoff FM, Thompson KD, Abdullah M

J Fish Dis, 2020 Oct;43(10):1249-1258.
PMID: 32830331 DOI: 10.1111/jfd.13222

Groupers are popular aquaculture species in South-East Asia, but their cultivation is affected by infectious disease outbreaks. Mucosa-associated lymphoid tissues provide a first-line defence against pathogens; however, few studies are available relating to cellular or proteomic responses of mucosal immunity in grouper. Skin, gill and intestine were sampled from brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) at 4 and 96 hr post-infection (hpi) and 7 days post-infection (dpi) following intraperitoneal infection with Vibrio harveyi, and stained with haematoxylin/eosin and Alcian Blue/periodic acid-Schiff. Skin mucus was analysed by 2D-gel electrophoresis, and proteins modulated by the bacterial infection identified. In the infected fish, significant increases in sacciform cells in skin and increased levels of nucleoside diphosphate kinase in mucus were detected at 4 hpi. At 96 hpi, goblet cells containing acidic mucins significantly increased in the intestine, while those containing mixed mucins increased in skin and gills of infected fish. Proteasome subunit alpha type-I and extracellular Cu/Zn superoxide dismutase levels also increased in mucus. Rodlet and mast cells did not appear to respond to the infection. Mucosal tissues of grouper appeared actively involved in response to Vibrio infection. This information may help future research on improving grouper health, production and vaccine development.
Fulltext An Early Reading Assessment Battery for Multilingual Learners in Malaysia

Lee JAC, Lee S, Yusoff NFM, Ong PH, Nordin ZS, Winskel H

Front Psychol, 2020;11:1700.
PMID: 32754104 DOI: 10.3389/fpsyg.2020.01700

The aim of the study was to develop a new comprehensive reading assessment battery for multi-ethnic and multilingual learners in Malaysia. Using this assessment battery, we examined the reliability, validity, and dimensionality of the factors associated with reading difficulties/disabilities in the Malay language, a highly transparent alphabetic orthography. In order to further evaluate the reading assessment battery, we compared results from the assessment battery with those obtained from the Malaysian national screening instrument. In the study, 866 Grade 1 children from multi-ethnic and multilingual backgrounds from 11 government primary schools participated. The reading assessment battery comprised 13 assessments, namely, reading comprehension, spelling, listening comprehension, letter name knowledge, letter name fluency, rapid automatized naming, word reading accuracy, word reading efficiency, oral reading fluency, expressive vocabulary, receptive vocabulary, elision, and phonological memory. High reliability and validity were found for the assessments. An exploratory factor analysis yielded three main constructs: phonological-decoding, sublexical-fluency, and vocabulary-memory. Phonological-decoding was found to be the most reliable construct that distinguished between at-risk and non-at-risk children. Identifying these underlying factors will be useful for detecting children at-risk for developing reading difficulties in the Malay language. In addition, these results highlight the importance of including a range of reading and reading-related measures for the early diagnosis of reading difficulties in this highly transparent orthography.
Interaction of monomeric Ebola VP40 protein with a plasma membrane: A coarse-grained molecular dynamics (CGMD) simulation study

Mohamad Yusoff MA, Abdul Hamid AA, Mohammad Bunori N, Abd Halim KB

J Mol Graph Model, 2018 Jun;82:137-144.
PMID: 29730487 DOI: 10.1016/j.jmgm.2018.04.010

Ebola virus is a lipid-enveloped filamentous virus that affects human and non-human primates and consists of several types of protein: nucleoprotein, VP30, VP35, L protein, VP40, VP24, and transmembrane glycoprotein. Among the Ebola virus proteins, its matrix protein VP40 is abundantly expressed during infection and plays a number of critical roles in oligomerization, budding and egress from the host cell. VP40 exists predominantly as a monomer at the inner leaflet of the plasma membrane, and has been suggested to interact with negatively charged lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS) via its cationic patch. The hydrophobic loop at the C-terminal domain has also been shown to be important in the interaction between the VP40 and the membrane. However, details of the molecular mechanisms underpinning their interactions are not fully understood. This study aimed at investigating the effects of mutation in the cationic patch and hydrophobic loop on the interaction between the VP40 monomer and the plasma membrane using coarse-grained molecular dynamics simulation (CGMD). Our simulations revealed that the interaction between VP40 and the plasma membrane is mediated by the cationic patch residues. This led to the clustering of PIP2 around the protein in the inner leaflet as a result of interactions between some cationic residues including R52, K127, K221, K224, K225, K256, K270, K274, K275 and K279 and PIP2 lipids via electrostatic interactions. Mutation of the cationic patch or hydrophobic loop amino acids caused the protein to bind at the inner leaflet of the plasma membrane in a different orientation, where no significant clustering of PIP2 was observed around the mutated protein. This study provides basic understanding of the interaction of the VP40 monomer and its mutants with the plasma membrane.
Self-sampling versus physician sampling for cervical cancer screening; The agreement of cytological diagnoses. [Abstract]

Mohamad Zaki FH, Nik Hussain NH, Ismail P, Wan Yusoff WZ, Othman NH

Pathology, 2016 Feb;48 Suppl 1:S148.
PMID: 27772923 DOI: 10.1016/j.pathol.2015.12.402

Background: The major problem with cervical cancer screening in countries which have no organized national screening program for cervical cancer is sub-optimal participation. Implementation of self-sampling method may increase the participation of women to screen for cervical cancer.
Aims: To determine the agreement of cytological diagnoses made on samples collected by women themselves (self-sampling) versus cytological diagnoses made on samples collected
by physicians (Physician sampling)
Methods: We invited women volunteers to undergo two procedures; cervical self-sampling using the Evalyn brush and physician scraping using Cervex brush. They women were
shown a video presentation on how to take their own cervical samples before the procedure. The samples taken by physicians were taken as per routine testing (Gold Standard). All
samples were subjected to Thin Prep monolayer smears. The diagnoses made were according to the Bethesda classification. The results from the two sampling methods were analysed and compared.
Results: A total of 367 women were recruited into the study. Thin Prep smears by physicians were better in terms of volume and variety of the cells seen. There is significant good agreement of the cytological diagnoses made on the samples from the two sampling methods with the Kappa value of 0.568 (p=0.040). The Thin Prep smears by self-sampling method were better in detecting microorganisms.
Conclusion: This study shows that samples taken by women themselves (self-sampling) and physicians sampling had good cytology agreement. Self-sampling could be the method of
choice in countries in which the coverage of women attending clinics for screening for cervical cancer is poor.
Construction of a novel inducible expression vector for Lactococcus lactis M4 and Lactobacillus plantarum Pa21

Maidin MS, Song AA, Jalilsood T, Sieo CC, Yusoff K, Rahim RA

Plasmid, 2014 Jul;74:32-8.
PMID: 24879963 DOI: 10.1016/j.plasmid.2014.05.003

A vector that drives the expression of the reporter gusA gene in both Lactobacillus plantarum and Lactococcus lactis was constructed in this study. This vector contained a newly characterized heat shock promoter (Phsp), amplified from an Enterococcus faecium plasmid, pAR6. Functionality and characterization of this promoter was initially performed by cloning Phsp into pNZ8008, a commercial lactococcal plasmid used for screening of putative promoters which utilizes gusA as a reporter. It was observed that Phsp was induced under heat, salinity and alkaline stresses or a combination of all three stresses. The newly characterized Phsp promoter was then used to construct a novel Lactobacillus vector, pAR1801 and its ability to express the gusA under stress-induced conditions was reproducible in both Lb. plantarum Pa21 and L. lactis M4 hosts.
Fulltext Draft Genome Sequence of a Vibrio parahaemolyticus Strain, KS17.S5-1, with Multiple Antibiotic Resistance Genes, Which Causes Acute Hepatopancreatic Necrosis Disease in Penaeus monodon in the West Coast of Peninsular Malaysia

Devadas S, Bhassu S, Christie Soo TC, Mohamed Iqbal SN, Yusoff FM, Shariff M

Microbiol Resour Announc, 2018 Jul;7(2).
PMID: 30533806 DOI: 10.1128/MRA.00829-18

We report the first draft genome sequence of a Vibrio parahaemolyticus strain (VpAHPND), which causes acute hepatopancreatic necrosis disease (AHPND) in Penaeus monodon. The strain has a pVA1-like plasmid carrying pirAvp and pirBvp genes. Whole-genome comparisons revealed >98% similarity to VpAHPND isolates from Thailand, Mexico, and Vietnam.
Fulltext Draft Genome Sequence of the Shrimp Pathogen Vibrio parahaemolyticus ST17.P5-S1, Isolated in Peninsular Malaysia

Devadas S, Bhassu S, Christie Soo TC, Yusoff FM, Shariff M

Microbiol Resour Announc, 2018 Sep;7(11).
PMID: 30533648 DOI: 10.1128/MRA.01053-18

We sequenced the genome of Vibrio parahaemolyticus strain ST17.P5-S1, isolated from Penaeus vannamei cultured in the east coast of Peninsular Malaysia. The strain contains several antibiotic resistance genes and a plasmid encoding the Photorhabdus insect-related (Pir) toxin-like genes, pirAvp and pirBvp, associated with acute hepatopancreatic necrosis disease (AHPND).
Prayer Moderating Job Stress Among Muslim Nursing Staff at the University of Malaya Medical Centre (UMMC)

Achour M, Muhamad A, Syihab AH, Mohd Nor MR, Mohd Yusoff MYZ

J Relig Health, 2021 Feb;60(1):202-220.
PMID: 31079348 DOI: 10.1007/s10943-019-00834-6

This study examines how Salat (prayer) in Islam moderates the relationship between job stress and life satisfaction among Muslim nursing staff. The researchers sampled 335 nursing staff employed at the University of Malaya Medical Centre in Kuala Lumpur, Malaysia. Their ages ranged from 21 to 60 years. The findings indicate the job stress was associated negatively with life satisfaction; there is a strong positive and significant correlation, respectively, between Salat and life satisfaction and that Salat reduces stress and increases the life satisfaction of Muslim nurses. Thus, Salat has a moderating effect on job stress and life satisfaction of Muslim nurses.
PF4-Induced apoptosis in breast cancer in vivo study: The role of the caspases family

Din TA, Seeni A, Yusoff MS, Shamsuddin S, Jaafar H

Pathology, 2016 Feb;48 Suppl 1:S122.
PMID: 27772839 DOI: 10.1016/j.pathol.2015.12.324
Using game controller as position tracking sensor for 3D freehand ultrasound imaging

Chan VS, Mohamed F, Yusoff YA, Dewi DEO, Anuar A, Shamsudin MA, et al.

Med Biol Eng Comput, 2020 May;58(5):889-902.
PMID: 31599379 DOI: 10.1007/s11517-019-02044-4

Position tracking has been widely used in medical applications, especially in 3D ultrasound imaging, where it has transformed the 2D slice limitation into 3D volume with bigger clinical impacts. As a game controller can also produce position tracking information, it has the potential to act as a low-cost and portable position tracker for ultrasound probes. This paper aims to investigate the feasibility of a game controller to perform as a position tracker and to design its implementation in 3D ultrasound imaging. The study consists of data acquisition and 3D ultrasound reconstruction for visualization. The data acquisition is accomplished by capturing the 2D ultrasound frame and its relative positional and orientation data by using an ultrasound probe and game controller respectively. These data are further reconstructed to produce 3D ultrasound volume for visualization. Our experiments include game controller position tracker testing and 3D ultrasound reconstruction on baby phantom. The results have confirmed that the game controller performance was closely aligned with that of in a robot arm. Also, the 3D ultrasound reconstruction implementation has revealed promising outcomes. With these features, the function of the currently available ultrasound probes can be prospectively improved using a game controller position tracker effectively. Graphical Abstract.
Dose dependent elevation of plasma tocotrienol levels and its effect on arterial compliance, plasma total antioxidant status, and lipid profile in healthy humans supplemented with tocotrienol rich vitamin E

Rasool AH, Yuen KH, Yusoff K, Wong AR, Rahman AR

J Nutr Sci Vitaminol (Tokyo), 2006 Dec;52(6):473-8.
PMID: 17330512

Tocotrienols are a class of vitamin E reported to be potent antioxidants, besides having the ability to inhibit the HMG-CoA reductase enzyme. This study assessed the effects of 3 doses of tocotrienol-rich vitamin E (TRE) on plasma tocotrienol isomer concentration, arterial compliance, plasma total antioxidant status (TAS), aortic systolic blood pressure (ASBP), serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) in healthy males.
METHODOLOGY: This randomised, blinded end-point, placebo-controlled clinical trial with a parallel design involved 36 healthy male subjects who took either an oral placebo or TRE at doses of 80, 160 or 320 mg daily for 2 mo. Baseline and end-of-treatment measurements of vitamin E concentration, arterial compliance [assessed by aortic femoral pulse wave velocity (PWV) and augmentation index (AI)], ASBP, plasma TAS, serum TC and LDL-C were taken.
RESULTS: Baseline tocotrienol isomer concentrations were low and not detectable in some subjects. Upon supplementation, all TRE-treated groups showed significant difference from placebo for their change in alpha, gamma and delta tocotrienol concentrations from baseline to end of treatment. There was a linear dose and blood level relationship for all the isomers. There was no significant difference between groups for their change in PWV, AI, plasma TAS, ASBP, TC or LDL-C from baseline to end of treatment. Groups 160 mg (p = 0.024) and 320 mg (p = 0.049) showed significant reductions in their ASBP. Group 320 mg showed a significant 9.2% improvement in TAS.
CONCLUSION: TRE at doses up to 320 mg daily were well tolerated. Treatment significantly increased alpha, delta, and gamma tocotrienol concentrations but did not significantly affect arterial compliance, plasma TAS, serum TC or LDL-C levels in normal subjects.
Fulltext Survival analysis of cholangiocarcinoma: a 10-year experience in Malaysia

Yusoff AR, Razak MM, Yoong BK, Vijeyasingam R, Siti ZM

World J Gastroenterol, 2012 Feb 7;18(5):458-65.
PMID: 22346252 DOI: 10.3748/wjg.v18.i5.458

To investigate the clinical features and survival of patients treated for cholangiocarcinoma in our institution and to analyze the factors affecting their survival.
Secretion of recombinant xylanase in Lactococcus lactis using signal peptides Usp45 and Spk1

Roslan AM, Mustafa Kamil A, Chandran C, Song AA, Yusoff K, Abdul Rahim R

Biotechnol Lett, 2020 Sep;42(9):1727-1733.
PMID: 32335791 DOI: 10.1007/s10529-020-02894-1

OBJECTIVE: The effect of two signal peptides, namely Usp45 and Spk1 on the secretion of xylanase in Lactococcus lactis was analysed.
RESULTS: Xylanase was successfully expressed in Lactococcus lactis. Recombinant xylanase fused to either signal peptide Usp45 or Spk1 showed halo zone on Remazol Brilliant Blue-Xylan plates. This indicated that the xylanase was successfully secreted from the cell. The culture supernatants of strains secreting the xylanase with help of the Spk1 and Usp45 signal peptides contained 49.7 U/ml and 34.4 U/ml of xylanase activity, respectively.
CONCLUSION: Although Usp45 is the most commonly used signal peptide when secreting heterologous proteins in Lactococcus lactis, this study shows that Spk1 isolated from Pediococcus pentosaceus was superior to Usp45 in regard to xylanase protein secretion.
Fulltext Antimicrobial activity and mode of action of terpene linalyl anthranilate against carbapenemase-producing Klebsiella pneumoniae

Yang SK, Yusoff K, Ajat M, Yap WS, Lim SE, Lai KS

J Pharm Anal, 2021 Apr;11(2):210-219.
PMID: 34012697 DOI: 10.1016/j.jpha.2020.05.014

Mining of plant-derived antimicrobials is the major focus at current to counter antibiotic resistance. This study was conducted to characterize the antimicrobial activity and mode of action of linalyl anthranilate (LNA) against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). LNA alone exhibited bactericidal activity at 2.5% (V/V), and in combination with meropenem (MPM) at 1.25% (V/V). Comparative proteomic analysis showed a significant reduction in the number of cytoplasmic and membrane proteins, indicating membrane damage in LNA-treated KPC-KP cells. Up-regulation of oxidative stress regulator proteins and down-regulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that LNA increases both bacterial surface charge and membrane permeability. Ethidium bromide influx/efflux assay showed increased uptake of ethidium bromide in LNA-treated cells, inferring membrane damage. Furthermore, intracellular leakage of nucleic acid and proteins was detected upon LNA treatment. Scanning and transmission electron microscopies again revealed the breakage of bacterial membrane and loss of intracellular materials. LNA was found to induce oxidative stress by generating reactive oxygen species (ROS) that initiate lipid peroxidation and damage the bacterial membrane. In conclusion, LNA generates ROS, initiates lipid peroxidation, and damages the bacterial membrane, resulting in intracellular leakage and eventually killing the KPC-KP cells.

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