Knowledge and attitudes of medical and pharmacy university students regarding monkeypox: a multicenter, cross-sectional study in Vietnam.

BACKGROUND: In May 2022, monkeypox (mpox) suddenly reappeared and rapidly spread worldwide. This global outbreak was a public health emergency of international concern. This study investigated Vietnamese medical and pharmacy university students' knowledge and attitudes towards mpox. METHODS: This online cross-section survey was administered among students in four universities using a convenience sampling method. A semi-structured questionnaire was shared among students via a Google Forms link (quick-response code) in university amphitheaters at recess. Frequency (percentage) and mean (standard division) were used for descriptive statistics. Univariate and multivariate linear regression analyses were conducted to identify factors associated with students' mpox knowledge and attitudes. A p-value < 0.001 was considered statistical significance. RESULTS: A total of 1,848 students participated in this study (including 1,030 medical students and 818 pharmacy students). Their common sources for seeking mpox/health information included the Internet (89.7%) and mass media (64.2%). Students' average knowledge and attitude scores were 11.542 ± 3.910 (range: 0-23) and 30.301 ± 3.738 (range: 9-45), respectively. The average knowledge score of pharmacy students (12.139 ± 3.545) was significantly higher than that of medical students (11.067 ± 4.118) (p < 0.001). The average attitude score of the former (30.454 ± 3.787) was comparatively higher than that of the latter (30.181 ± 3.696), but this difference was insignificant (p = 0.111). Factors significantly associated with students' mpox knowledge and/or attitudes were their sex, age, year of study, residence, major, grade point average, type of university, seeking mpox information in the past, and using reliable sources to seek health information (p < 0.001). A positive correlation existed between students' knowledge and attitude scores (p < 0.001). CONCLUSIONS: Students' knowledge about mpox was relatively poor, while their attitudes were comparatively positive. Universities and relevant authorities should focus on the above factors and use multiple platforms and channels of communication to guarantee that trustworthy information about mpox can reach as many students as possible.

Long-term SARS-CoV-2-specific immune and inflammatory responses in individuals recovering from COVID-19 with and without post-acute symptoms.

We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses, soluble markers of inflammation, and antibody levels and neutralization capacity longitudinally in 70 individuals with PCR-confirmed SARS-CoV-2 infection. Participants represent a spectrum of illness and recovery, including some with persistent viral shedding in saliva and many experiencing post-acute sequelae of SARS-CoV-2 infection (PASC). T cell responses remain stable for up to 9 months. Whereas the magnitude of early CD4+ T cell immune responses correlates with severity of initial infection, pre-existing lung disease is independently associated with higher long-term SARS-CoV-2-specific CD8+ T cell responses. Among participants with PASC 4 months following coronavirus disease 2019 (COVID-19) symptom onset, we observe a lower frequency of CD8+ T cells expressing CD107a, a marker of degranulation, in response to Nucleocapsid (N) peptide pool stimulation, and a more rapid decline in the frequency of N-specific interferon-γ-producing CD8+ T cells. Neutralizing antibody levels strongly correlate with SARS-CoV-2-specific CD4+ T cell responses.

SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay.

Interpretation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveillance studies is limited by poorly defined performance of antibody assays over time in individuals with different clinical presentations. We measured antibody responses in plasma samples from 128 individuals over 160 days using 14 assays. We found a consistent and strong effect of disease severity on antibody magnitude, driven by fever, cough, hospitalization, and oxygen requirement. Responses to spike protein versus nucleocapsid had consistently higher correlation with neutralization. Assays varied substantially in sensitivity during early convalescence and time to seroreversion. Variability was dramatic for individuals with mild infection, who had consistently lower antibody titers, with sensitivities at 6 months ranging from 33 to 98% for commercial assays. Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on infection severity, timing, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.

SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay.

Serosurveillance studies are critical for estimating SARS-CoV-2 transmission and immunity, but interpretation of results is currently limited by poorly defined variability in the performance of antibody assays to detect seroreactivity over time in individuals with different clinical presentations. We measured longitudinal antibody responses to SARS-CoV-2 in plasma samples from a diverse cohort of 128 individuals over 160 days using 14 binding and neutralization assays. For all assays, we found a consistent and strong effect of disease severity on antibody magnitude, with fever, cough, hospitalization, and oxygen requirement explaining much of this variation. We found that binding assays measuring responses to spike protein had consistently higher correlation with neutralization than those measuring responses to nucleocapsid, regardless of assay format and sample timing. However, assays varied substantially with respect to sensitivity during early convalescence and in time to seroreversion. Variations in sensitivity and durability were particularly dramatic for individuals with mild infection, who had consistently lower antibody titers and represent the majority of the infected population, with sensitivities often differing substantially from reported test characteristics (e.g., amongst commercial assays, sensitivity at 6 months ranged from 33% for ARCHITECT IgG to 98% for VITROS Total Ig). Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on the severity of the initial infection, timing relative to infection, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.

Long-Term SARS-CoV-2-Specific Immune and Inflammatory Responses Across a Clinically Diverse Cohort of Individuals Recovering from COVID-19.

A detailed understanding of long-term SARS-CoV-2-specific T cell responses and their relationship to humoral immunity and markers of inflammation in diverse groups of individuals representing the spectrum of COVID-19 illness and recovery is urgently needed. Data are also lacking as to whether and how adaptive immune and inflammatory responses differ in individuals that experience persistent symptomatic sequelae months following acute infection compared to those with complete, rapid recovery. We measured SARS-CoV-2-specific T cell responses, soluble markers of inflammation, and antibody levels and neutralization capacity longitudinally up to 9 months following infection in a diverse group of 70 individuals with PCR-confirmed SARS-CoV-2 infection. The participants had varying degrees of initial disease severity and were enrolled in the northern California Long-term Impact of Infection with Novel Coronavirus (LIINC) cohort. Adaptive T cell responses remained remarkably stable in all participants across disease severity during the entire study interval. Whereas the magnitude of the early CD4+ T cell immune response is determined by the severity of initial infection (participants requiring hospitalization or intensive care), pre-existing lung disease was significantly associated with higher long-term SARS-CoV2-specific CD8+ T cell responses, independent of initial disease severity or age. Neutralizing antibody levels were strongly correlated with SARS-CoV-2-specific CD4+ T but not CD8+ T cell responses. Importantly, we did not identify substantial differences in long-term virus-specific T cell or antibody responses between participants with and without COVID-19-related symptoms that persist months after initial infection.

Toward Shared Working Space of Human and Robotic Agents Through Dipole Flow Field for Dependable Path Planning.

Recent industrial developments in autonomous systems, or agents, which assume that humans and the agents share the same space or even work in close proximity, open for new challenges in robotics, especially in motion planning and control. In these settings, the control system should be able to provide these agents a reliable path following control when they are working in a group or in collaboration with one or several humans in complex and dynamic environments. In such scenarios, these agents are not only moving to reach their goals, i.e., locations, they are also aware of the movements of other entities to find a collision-free path. Thus, this paper proposes a dependable, i.e., safe, reliable and effective, path planning algorithm for a group of agents that share their working space with humans. Firstly, the method employs the Theta* algorithm to initialize the paths from a starting point to a goal for a set of agents. As Theta* algorithm is computationally heavy, it only reruns when there is a significant change of the environment. To deal with the movements of the agents, a static flow field along the configured path is defined. This field is used by the agents to navigate and reach their goals even if the planned trajectories are changed. Secondly, a dipole field is calculated to avoid the collision of agents with other agents and human subjects. In this approach, each agent is assumed to be a source of a magnetic dipole field in which the magnetic moment is aligned with the moving direction of the agent. The magnetic dipole-dipole interactions between these agents generate repulsive forces to help them to avoid collision. The effectiveness of the proposed approach has been evaluated with extensive simulations. The results show that the static flow field is able to drive agents to the goals with a small number of requirements to update the path of agents. Meanwhile, the dipole flow field plays an important role to prevent collisions. The combination of these two fields results in a safe path planning algorithm, with a deterministic outcome, to navigate agents to their desired goals.

Pyrimidoaminotropanes as potent, selective, and efficacious small molecule kinase inhibitors of the mammalian target of rapamycin (mTOR).

We have recently reported a series of tetrahydroquinazoline (THQ) mTOR inhibitors that produced a clinical candidate 1 (GDC-0349). Through insightful design, we hoped to discover and synthesize a new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable, and efficacious mTOR inhibitor.

Discovery and Biological Profiling of Potent and Selective mTOR Inhibitor GDC-0349.

Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.

Potent, selective, and orally bioavailable inhibitors of the mammalian target of rapamycin kinase domain exhibiting single agent antiproliferative activity.

Selective inhibitors of mammalian target of rapamycin (mTOR) kinase based upon saturated heterocycles fused to a pyrimidine core were designed and synthesized. Each series produced compounds with K(i) < 10 nM for the mTOR kinase and >500-fold selectivity over closely related PI3 kinases. This potency translated into strong pathway inhibition, as measured by phosphorylation of mTOR substrate proteins and antiproliferative activity in cell lines with a constitutively active PI3K pathway. Two compounds exhibiting suitable mouse PK were profiled in in vivo tumor models and were shown to suppress mTORC1 and mTORC2 signaling for over 12 h when dosed orally. Both compounds were additionally shown to suppress tumor growth in vivo in a PC3 prostate cancer model over a 14 day study.

Novel method to assess antiretroviral target trough concentrations using in vitro susceptibility data.

Durable suppression of HIV-1 replication requires the establishment of antiretroviral drug concentrations that exceed the susceptibility of the virus strain(s) infecting the patient. Minimum plasma drug concentrations (C(trough)) are correlated with response, but determination of target C(trough) values is hindered by a paucity of in vivo concentration-response data. In the absence of these data, in vitro susceptibility measurements, adjusted for serum protein binding, can provide estimations of suppressive in vivo drug concentrations. We derived serum protein binding correction factors (PBCF) for protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and an integrase inhibitor by measuring the effect of a range of human serum concentrations on in vitro drug susceptibility measured with the PhenoSense HIV assay. PBCFs corresponding to 100% HS were extrapolated using linear regression and ranged from 1.4 for nevirapine to 77 for nelfinavir. Using the mean 95% inhibitory concentration (IC(95)) for ≥1,200 drug-susceptible viruses, we calculated protein-bound IC(95) (PBIC(95)) values. PBIC(95) values were concordant with the minimum effective C(trough) values that were established in well-designed pharmacodynamic studies (e.g., indinavir, saquinavir, and amprenavir). In other cases, the PBIC(95) values were notably lower (e.g., darunavir, efavirenz, and nevirapine) or higher (nelfinavir and etravirine) than existing target recommendations. The establishment of PBIC(95) values as described here provides a convenient and standardized approach for estimation of the minimum drug exposure that is required to maintain viral suppression and prevent the emergence of drug-resistant variants, particularly when in vivo concentration-response relationships are lacking.

Potent, selective, and orally bioavailable inhibitors of mammalian target of rapamycin (mTOR) kinase based on a quaternary substituted dihydrofuropyrimidine.

A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i) < 1.0 nM and were highly (>100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.

Sensitivity of phenotypic susceptibility analyses for nonthymidine nucleoside analogues conferred by K65R or M184V in mixtures with wild-type HIV-1.

Thymidine-sparing triple-nucleoside regimens have exhibited poor virologic response despite apparent phenotypic susceptibility to 2 of 3 regimen components at early time points. Phenotypic resistance masking by wild-type virus may explain this discrepancy.Consistent with this notion were (1) the presence of low level nucleoside reverse-transcriptase inhibitor-resistant human immunodeficiency virus in subjects receiving failing first-line regimens consisting of tenofovir (TDF), abacavir (ABC), and lamivudine (3TC); (2) lower fold resistance associated with mixtures versus mutants in a clinical-isolate database; and (3) dose dependent changes in susceptibility to ABC, 3TC, TDF, and didanosine on titration of K65R and/or M184V with wild-type virus. These findings underscore the limitations of stand-alone phenotypic susceptibility measures and emphasize the importance of complementary and/or more sensitive techniques.

Synthesis of glutamic acid analogs as potent inhibitors of leukotriene A4 hydrolase.

Leukotriene B(4) (LTB(4)) is a potent pro-inflammatory mediator that has been implicated in the pathogenesis of multiple diseases, including psoriasis, inflammatory bowel disease, multiple sclerosis and asthma. As a method to decrease the level of LTB(4) and possibly identify novel treatments, inhibitors of the LTB(4) biosynthetic enzyme, leukotriene A(4) hydrolase (LTA(4)-h), have been explored. Here we describe the discovery of a potent inhibitor of LTA(4)-h, arylamide of glutamic acid 4f, starting from the corresponding glycinamide 2. Analogs of 4f are then described, focusing on compounds that are both active and stable in whole blood. This effort culminated in the identification of amino alcohol 12a and amino ester 6b which meet these criteria.

The ubiquitin-conjugating enzyme E2-EPF is overexpressed in primary breast cancer and modulates sensitivity to topoisomerase II inhibition.

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER(-) MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G(2)/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G(2) checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIalpha protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness.

Solid-phase synthesis of naphthylamidines as factor VIIa/tissue factor inhibitors.

Reductive amination followed by acylation of polymer-linked formyl aryl amidines generate combinatorial libraries of aryl amidines 8-13. Potent small molecule naphthylamidine inhibitors 12 (Ki<100 nM) of FVIIa/TF have been discovered and their activity against other serine proteases in the coagulation cascade is reported.

Discovery and characterization of a potent and selective non-amidine inhibitor of human factor Xa.

Benzothiophene-anthranilamide 1 (3-chloro-N-[2-[[(4-fluorophenyl)amino]carbonyl]-4-methylphenyl]benzo[b]thiophene-2-carboxamide) was discovered by high throughput screening to be a highly potent and selective non-amidine inhibitor of human factor Xa with a K(i) of 15+/-4nM. Compound 1 is a selective inhibitor of human factor Xa as suggested by the K(i)((app)) determined for nine other human serine proteases and bovine trypsin. The activity of reconstituted human prothrombinase complex was inhibited by compound 1 when assayed in physiological concentrations of the substrate prothrombin. However, 27-fold higher inhibitor concentrations were needed to achieve the same level of inhibition than were required for the inhibition of free factor Xa, due in part to non-specific binding of the inhibitor to phospholipid under the assay conditions. Failure to demonstrate enzymatic cleavage of compound 1 suggests that compound 1 is solely an inhibitor rather than a substrate for factor Xa. The inhibition of factor Xa by compound 1 was reversible upon dilution of the enzyme/inhibitor mixture. Analyses of the inhibition mechanism with Dixon, Cornish-Bowden, and Lineweaver-Burk plots showed that compound 1 is a linear mixed-type inhibitor with 5-fold higher affinity for free factor Xa than the factor Xa/substrate complex. The linear mixed-type inhibition suggests that compound 1 binds to the active site region of factor Xa, but its binding cannot be fully displaced by the substrate S2222 (1:1 mixture of N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide and N-benzoyl-Ile-Glu(gamma-OMe)-Gly-Arg-p-nitroanilide hydrochloride). Thus, the inhibition mechanism for compound 1 is novel compared to most serine protease inhibitors including amidine-containing factor Xa inhibitors, which rely on binding to the S1 pocket of the enzyme active site. Compound 1 represents an attractive, novel structural template for further development of efficacious, safe, and potentially orally active human factor Xa inhibitors.

Structure-activity relationships of substituted benzothiophene-anthranilamide factor Xa inhibitors.

Compound 1 was identified by high throughput screening as a novel, potent, non-amidine factor Xa inhibitor with good selectivity against thrombin and trypsin. A series of modifications of the three aromatic groups of 1 was investigated. Substitution of chlorine or bromine for fluorine on the aniline ring led to the discovery of subnanomolar factor Xa inhibitors. Positions on the anthranilic acid ring that can accommodate further substitution were also identified.

Design, synthesis, and activity of a novel series of factor Xa inhibitors: optimization of arylamidine groups.

A novel series of diaryloxypyridines have been designed as selective nanomolar factor Xa (fXa) inhibitors for use as anticoagulants. In this paper, we describe our efforts to identify an additional interaction and a replacement for the distal amidine group that binds in the S3/S4 pocket of fXa. Introduction of a hydroxyl group para to the proximal amidine group increases the potency vs fXa by 1-2 orders of magnitude, which is the result of a hydrogen bond to Ser195 of the catalytic triad. A methyl imidazoline and a dimethylamide are good alternatives for the second amidine. These substitutions have increased the selectivity vs the related serine proteases trypsin and thrombin. The synthesis, in vitro activity, and hypothetical modes of binding to fXa based on trypsin crystallographic data are outlined.

Design and synthesis of aminophenol-based factor Xa inhibitors.

A novel potent and selective aminophenol scaffold for fXa inhibitors was developed from a previously reported benzimidazole-based naphthylamidine template. The aminophenol template is more synthetically accessible than the benzimidazole template, which simplified the introduction of carboxylic acid groups. Substitution of a propenyl-para-hydroxy-benzamidine group on the aminophenol template produced selective, sub-nanomolar fXa inhibitors. The potency of the inhibitors is partially explained with the aid of a trypsin complex crystal structure.

Benzimidazole-based fXa inhibitors with improved thrombin and trypsin selectivity.

Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the 4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus thrombin, but selectivity versus trypsin as well.