A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity.

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays, including gene expression analysis, protein-protein interaction, and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remain elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in living cells and in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in live cell and in vitro assays performed using lysates prepared from cells expressing the proteins and with the furimazine substrate. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity. SIGNIFICANCE: Bioluminescence has been extensively utilized in developing a variety of biological and biomedical assays. In this regard, engineering of brighter bioluminescent proteins, i.e. luciferases, has played a significant role. This is acutely exemplified by the engineering of the NLuc luciferase, which is small in size and displays much enhanced bioluminescence and thermal stability compared to previously available luciferases. While enhanced bioluminescent activity is desirable in a multitude of biological and biomedical assays, it would also be useful to develop variants of the protein that display a prolonged bioluminescence activity. This is specifically relevant in designing assays that require bioluminescence for extended periods, such as in the case of biosensors designed for monitoring slow enzymatic or cellular signaling reactions, without necessitating multiple rounds of luciferase substrate addition or any specialized reagents that result in increased assay costs. In the current manuscript, we report a mutant NLuc that possesses a stable and prolonged bioluminescence activity, albeit lower than the wild-type NLuc, and envisage a wider application of the mutant NLuc in designing biosensors for monitoring slower biological and biomedical events.

Attitudes, motivators, and barriers toward influenza vaccination for children: a study from a conflict-ridden country.

BACKGROUND: Despite the increased recommendations for influenza vaccination, particularly among high-risk groups such as young children, Yemen lacks an influenza vaccination program, and the influenza vaccine is not included in the national immunization regime. This is exacerbated by the country's fragile infrastructure, as well as the devastating consequences of the ongoing conflict, which include child undernutrition and strained healthcare resources. Thus, the objective of the current study is to assess the public attitudes and perceptions toward vaccinating children against influenza in Yemen. METHODS: A cross-sectional study was conducted by distributing a validated survey questionnaire to potential participants using convenience sampling. Descriptive statistics were used to summarize sociodemographic data, knowledge of influenza vaccines, and attitudes and perceptions regarding vaccinating children against influenza. Logistic regression analysis was employed to identify associations between independent variables and the acceptance of vaccines for children. RESULTS: A total of 853 eligible individuals, parents and non-parents, successfully completed the survey. The uptake of the influenza vaccine among the participants was notably low as the majority (69.2%) had not previously received the vaccine, although the majority expressed a willingness to get vaccinated in the future (59.4%). The majority (68.5%) were willing to vaccinate children. The largest percentage of the participants who expressed hesitancy toward children's vaccination cited multiple reasons to reject the vaccine (39.7%), with concerns regarding the safety of the vaccine being the predominant barrier to its acceptance for children (29.6%). On the other hand, motivating factors for vaccinating children included the validation of the vaccine's safety and efficacy, endorsement of the vaccine by the government and physicians, integration of the vaccine into the national immunization program, and the provision of the vaccine free of charge and through schools. Significant predictors for vaccine acceptance in children included male gender, knowledge of the protective effect of the influenza vaccine, previous receipt of the vaccine, and a willingness to receive the vaccine in the future. CONCLUSIONS: The study highlights the need for educational health campaigns to raise awareness and remove misconceptions regarding influenza and the role, benefits, and availability of its vaccine. These findings can serve as a robust foundation for the future design and implementation of an influenza vaccination program for children in Yemen.

Functional Characterization of Novel MC4R Variants Identified in Two Unrelated Patients with Morbid Obesity in Qatar.

The leptin-melanocortin pathway is pivotal in appetite and energy homeostasis. Pathogenic variants in genes involved in this pathway lead to severe early-onset monogenic obesity (MO). The MC4R gene plays a central role in leptin-melanocortin signaling, and heterozygous variants in this gene are the most common cause of MO. A targeted gene panel consisting of 52 obesity-related genes was used to screen for variants associated with obesity. Variants were analyzed and filtered to identify potential disease-causing activity and validated using Sanger sequencing. We identified two novel heterozygous variants, c.253A>G p.Ser85Gly and c.802T>C p.Tyr268His, in the MC4R gene in two unrelated patients with morbid obesity and evaluated the functional impact of these variants. The impact of the variants on the MC4R gene was assessed using in silico prediction tools and molecular dynamics simulation. To further study the pathogenicity of the identified variants, GT1-7 cells were transfected with plasmid DNA encoding either wild-type or mutant MC4R variants. The effects of allelic variations in the MC4R gene on cAMP synthesis, MC4R protein level, and activation of PKA, ERB, and CREB signaling pathways in both stimulated and unstimulated ɑ-MSH paradigms were determined for their functional implications. In silico analysis suggested that the variants destabilized the MC4R structure and affected the overall dynamics of the MC4R protein, possibly leading to intracellular receptor retention. In vitro analysis of the functional impact of these variants showed a significant reduction in cell surface receptor expression and impaired extracellular ligand binding activity, leading to reduced cAMP production. Our analysis shows that the variants do not affect total protein expression; however, they are predicted to affect the post-translational localization of the MC4R protein to the cell surface and impair downstream signaling cascades such as PKA, ERK, and CREB signaling pathways. This finding might help our patients to benefit from the novel therapeutic advances for monogenic forms of obesity.

Identification of a potential DNA methyltransferase (DNMT) inhibitor.

DNA methyltransferases (DNMTs) play an important role in the epigenetic regulation of gene expression through the methylation of DNA. Since hypermethylation and consequent suppression of tumor suppressor genes are associated with cancer development and progression, DNA hypomethylating agents (HMAs) such as DNMT inhibitors have been proposed for cancer therapy. Two nucleoside analogues approved for the treatment of hematological cancers, decitabine and azacytidine, have poor pharmacokinetic properties, and hence there is a critical need for identifying novel HMAs. Virtual screening of a library of ∼40,000 compounds from the ZINC database, followed by molecular docking of 4,000 compounds having potential druggable properties with DNMT1, DNMT3A and DNMT3B were performed. One unique inhibitor (ZINC167686681) was identified that successfully passed through the Lipinski Rule of 5, geometry constraints as well as ADME/Tox filters and having strong binding energy for DNMTs. Further, molecular dynamics simulations of the docked complexes showed detailed structural features critical for its binding with the DNMTs and the stability of their interaction. Our study identified a compound with potential druggable properties and predicted to bind and inhibit DNMTs. Further investigations involving cellular and animal models of ZINC167686681 will help in potentially taking it into clinical trials for the treatment of cancers.Communicated by Ramaswamy H. Sarma.

Attitudes Toward Providing Open Access for Use of Biospecimens and Health Records: A Cross-Sectional Study from Jordan.

Purpose: Biospecimen repositories and big data generated from clinical research are critically important in advancing patient-centered healthcare. However, ethical considerations arising from reusing clinical samples and health records for subsequent research pose a hurdle for big-data health research. This study aims to assess the public's opinions in Jordan toward providing blanket consent for using biospecimens and health records in research. Participants and Methods: A cross-sectional study utilizing a self-reported questionnaire was carried out in different cities in Jordan, targeting adult participants. Outcome variables included awareness of clinical research, participation in clinical research, and opinions toward providing open access to clinical samples and records for research purposes. Descriptive analysis was utilized for reporting the outcome as frequency (percentages) out of the total responses. Univariate and multivariate logistic regression were used to investigate the association between independent variables and the outcome of interest. Results: A total of 1033 eligible participants completed the questionnaire. Although the majority (90%) were aware of clinical research, only 24% have ever participated in this type of research. About half (51%) agreed on providing blanket consent for the use of clinical samples, while a lower percentage (43%) agreed on providing open access to their health records. Privacy concerns and lack of trust in the researcher were cited as major barriers to providing blanket consent. Participation in clinical research and having health insurance were predictors for providing open access to clinical samples and records. Conclusion: The lack of public trust in Jordan toward data privacy is evident from this study. Therefore, a governance framework is needed to raise and maintain the public's trust in big-data research that warrants the future reuse of clinical samples and records. As such, the current study provides valuable insights that will inform the design of effective consent protocols required in data-intensive health research.

Knowledge, attitudes, perceptions, and practice toward seasonal influenza and its vaccine: A cross-sectional study from a country of conflict.

Background: The seasonal influenza vaccine is an important preventive measure against influenza and its associated complications. In Yemen, there is no seasonal influenza vaccination policy, and the influenza vaccine is excluded from the national immunization program. Data on vaccination coverage remain scarce with no previous surveillance programs or awareness campaigns implemented in the country. The current study aims to assess the awareness, knowledge, and attitudes of the public in Yemen toward seasonal influenza and their motivators and perceived barriers to receiving its vaccine. Methods: A cross-sectional survey was carried out using a self-administered questionnaire that was distributed to eligible participants using convenience sampling. Results: A total of 1,396 participants completed the questionnaire. The respondents showed a median knowledge score of influenza of 11.0/15.0, and most of them (70%) were able to recognize its modes of transmission. However, only 11.3% of the participants reported receiving the seasonal influenza vaccine. Physicians were the respondents' most preferred information source for influenza (35.2%), and their recommendation (44.3%) was the most cited reason for taking its vaccine. On the contrary, not knowing about the vaccine's availability (50.1%), concerns regarding the safety of the vaccine (17%), and not considering influenza as a threat (15.9%) were the main reported barriers to getting vaccinated. Conclusion: The current study showed a low uptake of influenza vaccines in Yemen. The physician's role in promoting influenza vaccination seems to be essential. Extensive and sustained awareness campaigns would likely increase the awareness of influenza and remove misconceptions and negative attitudes toward its vaccine. Equitable access to the vaccine can be promoted by providing it free of charge to the public.

Reversal of the unique Q493R mutation increases the affinity of Omicron S1-RBD for ACE2.

The SARS-CoV-2 Omicron variant containing 15 mutations, including the unique Q493R, in the spike protein receptor binding domain (S1-RBD) is highly infectious. While comparison with previously reported mutations provide some insights, the mechanism underlying the increased infections and the impact of the reversal of the unique Q493R mutation seen in BA.4, BA.5, BA.2.75, BQ.1 and XBB lineages is not yet completely understood. Here, using structural modelling and molecular dynamics (MD) simulations, we show that the Omicron mutations increases the affinity of S1-RBD for ACE2, and a reversal of the unique Q493R mutation further increases the ACE2-S1-RBD affinity. Specifically, we performed all atom, explicit solvent MD simulations using a modelled structure of the Omicron S1-RBD-ACE2 and compared the trajectories with the WT complex revealing a substantial reduction in the Cα-atom fluctuation in the Omicron S1-RBD and increased hydrogen bond and other interactions. Residue level analysis revealed an alteration in the interaction between several residues including a switch in the interaction of ACE2 D38 from S1-RBD Y449 in the WT complex to the mutated R residue (Q493R) in Omicron complex. Importantly, simulations with Revertant (Omicron without the Q493R mutation) complex revealed further enhancement of the interaction between S1-RBD and ACE2. Thus, results presented here not only provide insights into the increased infectious potential of the Omicron variant but also a mechanistic basis for the reversal of the Q493R mutation seen in some Omicron lineages and will aid in understanding the impact of mutations in SARS-CoV-2 evolution.

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression.

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein ß2-microglobulin (ß2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that down-regulate MHC-I expression to avoid CD8+ T cell recognition. Here, we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, down-regulate MHC-I expression using distinct mechanisms. First, ORF3a, a viroporin, reduces the global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a specific mechanism that allows immune evasion by SARS-CoV-2.

Corrigendum: Decreased interfacial dynamics caused by the N501Y mutation in the SARS-CoV-2 S1 spike:ACE2 complex.

[This corrects the article DOI: 10.3389/fmolb.2022.846996.].

Perceptions of Pharmacy Graduate Students Toward Research Ethics Education: A Cross-Sectional Study from a Developing Country.

Despite the potential value of graduate-level research ethics training, most Middle East countries, including Jordan, do not routinely offer formal research ethics training. In students enrolled in Jordanian master's level graduate program in pharmacy, the current study assessed: 1- differences in pre- and post-enrollment exposure to research ethics core themes, 2- whether this exposure was through a formal course or in an informal setting, and 3- student attitudes towards research ethics education and the need for integrating a dedicated research ethics course into pharmacy graduate programs. A 12-item on-line survey was developed by the authors and disseminated to a convenience sample of current and former master-level pharmacy students in Jordan. A total of 61 eligible respondents completed the survey. A minority of respondents (38%) acknowledged receiving research ethics training prior to enrollment into a postgraduate pharmacy program with nearly half (16%) describing this training as informal. In comparison, a larger percentage of the total respondents (56%) had received research ethics training during their postgraduate program enrollment, with nearly half of those (25%) indicating that this training was informal. A majority of respondents reported a strong need for integrating a formal research ethics course into postgraduate pharmacy curriculum (90%) to support their research training and thesis writing (89%). Overall, the study revealed a notable lack of research ethics education for graduate-level pharmacy students in Jordan.

A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor.

The main protease, Mpro, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting Mpro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with Mpro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in Mpro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

Decreased Interfacial Dynamics Caused by the N501Y Mutation in the SARS-CoV-2 S1 Spike:ACE2 Complex.

Coronavirus Disease of 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a massive health crisis across the globe, with some genetic variants gaining enhanced infectivity and competitive fitness, and thus significantly aggravating the global health concern. In this regard, the recent SARS-CoV-2 alpha, beta, and gamma variants (B.1.1.7, B.1.351, and P.1 lineages, respectively) are of great significance in that they contain several mutations that increase their transmission rates as evident from clinical reports. By the end of March 2021, these variants were accounting for about two-thirds of SARS-CoV-2 variants circulating worldwide. Specifically, the N501Y mutation in the S1 spike receptor binding domain (S1-RBD) of these variants have been reported to increase its affinity for ACE2, although the basis for this is not entirely clear yet. Here, we dissect the mechanism underlying the increased binding affinity of the N501Y mutant for ACE2 using molecular dynamics (MD) simulations of the available ACE2-S1-RBD complex structure (6M0J) and show a prolonged and stable interfacial interaction of the N501Y mutant S1-RBD with ACE2 compared to the wild type S1-RBD. Additionally, we find that the N501Y mutant S1-RBD displays altered dynamics that likely aids in its enhanced interaction with ACE2. By elucidating a mechanistic basis for the increased affinity of the N501Y mutant S1-RBD for ACE2, we believe that the results presented here will aid in developing therapeutic strategies against SARS-CoV-2 including designing of therapeutic agents targeting the ACE2-S1-RBD interaction.

Motivators and barriers towards clinical research participation: A population-based survey from an Arab MENA country.

Jordan was the first Arab country to enact clinical research regulations. The country has a well-flourished pharmaceutical industry that leans heavily on clinical research (CR) for drug development and post-marketing surveillance. In this cross-sectional study, we sought to assess the public's awareness and attitude towards CR as well as their perceived motivators and barriers to CR participation. A population-based, self-administered questionnaire was distributed to the general public in Jordan. Among the 1061 participants in this survey, 74% reported being aware of CR. The majority (70%) agreed to the role of CR in health promotion. Online information and healthcare staff were the two main sources of CR information for the participants. About 25% of the participants received prior invitations to participate in CR with 21% agreeing to participate. However, most participants of the current study (63%) were willing to participate in future CR. Contributing to science, benefiting others, and promoting one's own health were the top motivating factors for participating in CR; while time constraints, fear of research procedure, and lack of interest were the most cited reasons for rejecting participation. Filling out questionnaire surveys, donating blood samples, and participating in physical examinations were the main CR contributions of the participants. Nearly 31% of the participants believed that CR is conducted in a responsible and ethical manner, while 57% did not have an opinion regarding the same matter. In addition, 49% and 44% were neutral with regards to the degree of harm and confidentiality posed by CR. While only 27% disagreed that CR exposes participants to some form of harm, 48% either strongly agreed (15%) or agreed (33%) that it maintains high level of confidentiality for participants. The current study provides insight into the public's perception of CR in Jordan as well as its motivating factors and perceived barriers towards participating in CR. We envisage to utilize this insight as an aid in the design of vigilant future awareness campaigns and recruitment strategies.

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression.

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein ß 2 -microglobulin (ß 2 m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8 + T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8 + T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß 2 m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2. Significance Statement: Viruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with ß 2 m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of ß 2 m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.

Assessment of research ethics education offerings of pharmacy master programs in an Arab nation relative to top programs worldwide: A qualitative content analysis.

The importance of research ethics (RE) training has led academic and funding institutions to require that students, trainees, and faculty obtain such training at various stages of their careers. Despite the increasing awareness of the value RE education offers, this training requirement is absent in Jordan. We aimed to assess RE education offerings of pharmacy master programs in Jordan and compare with the top-ranked pharmacy graduate programs globally. Therefore, a list of universities that offer research-based pharmacy master programs was created. Each program was evaluated for the inclusion of RE education. A qualitative content analysis approach based on inductive reasoning and latent analysis was followed to analyze the data. Results of the study showed a lack of appropriate RE education for graduate-level pharmacy programs in Jordan with only 40% of the programs partially discuss selected topics related to RE. Regarding pharmacy graduate programs globally, 10% offer a standalone RE course, 40% offer some discussions related to RE, another 10% do not offer RE education in any form, and the remaining 40% of the programs were difficult to assess due to lack of sufficient information available online. Based on the findings of this study, training in RE is tends to be lacking in pharmacy graduate programs in Jordan and globally, with a greater lack in Jordan than globally. There is a need to incorporate formal RE education into programs that do not offer this type of instruction. Programs that formally touch on some aspects of RE need to expand the scope of topics to include more RE-related themes. Integrating a standalone RE course into pharmacy graduate programs is highly encouraged.

Phosphodiesterase 5 (PDE5): Structure-function regulation and therapeutic applications of inhibitors.

Phosphodiesterase 5 (PDE5) is one of the most well-studied phosphodiesterases (PDEs) that specifically targets cGMP typically generated by nitric oxide (NO)-mediated activation of the soluble guanylyl cyclase. Given the crucial role of cGMP generated through the activation of this cellular signaling pathway in a variety of physiologically processes, pharmacological inhibition of PDE5 has been demonstrated to have several therapeutic applications including erectile dysfunction and pulmonary arterial hypertension. While they are designed to inhibit PDE5, the inhibitors show different affinities and specificities against all PDE subtypes. Additionally, they have been shown to induce allosteric structural changes in the protein. These are mostly attributed to their chemical structure and, therefore, binding interactions with PDE catalytic domains. Therefore, understanding how these inhibitors interact with PDE5 and the structural basis of their selectivity is critically important for the design of novel, highly selective PDE5 inhibitors. Here, we review the structure of PDE5, how its function is regulated, and discuss the clinically available inhibitors that target phosphodiesterase 5, aiming to better understand the structural bases of their affinity and specificity. We also discuss the therapeutic indications of these inhibitors and the potential of repurposing for a wider range of clinical applications.

In Vitro Comparison of Antibacterial and Antibiofilm Activities of Selected Fluoroquinolones against Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus.

An in vitro overview of the inhibitory effects of selected fluoroquinolones against planktonic and biofilm cells of the methicillin-resistant Staphylococcus aureus (MRSA) strain American type culture collection (ATCC) 43300 and the Pseudomonas aeruginosa strain ATCC 27853 was carried out. Biofilm cells of both strains were less susceptible to the selected antibiotics than their planktonic counterparts. In addition, certain antibiotics were more effective against biofilm cells, while others performed better on the planktonic cells. Against P. aeruginosa, ciprofloxacin was the most potent on both planktonic and biofilm cells, whereas ofloxacin was the least potent on both biofilm and planktonic cells. Moxifloxacin and gatifloxacin were the most potent against both planktonic and biofilm MRSA bacteria, however, not in the same order of activity. Norfloxacin was the least active when tested against both planktonic and biofilm cells. The results of this work are expected to provide insight into the efficacy of various fluoroquinolones against MRSA and Pseudomonas aeruginosa biofilms. This study could form the basis for future clinical studies that could recommend special guidelines for the management of infections that are likely to involve bacteria in their biofilm state.