Repurposing anti-cancer porphyrin derivative drugs to target SARS-CoV-2 envelope.

Antiviral medicines to treat COVID-19 are still scarce. Porphyrins and porphyrin derivatives (PDs) usually present broad-spectrum antiviral activity with low risk of resistance development. In fact, some PDs are clinically approved to be used in anti-cancer photodynamic therapy and repurposing clinically approved PDs might be an alternative to treat COVID-19. Here, we characterize the ability of temoporfin, verteporfin, talaporfin and redaporfin to inactivate SARS-CoV-2 infectious particles. PDs light-dependent and -independent effect on SARS-CoV-2 infectivity were evaluated. PDs photoactivation successfully inactivated SARS-CoV-2 with very low concentrations and light dose. However, only temoporfin and verteporfin inactivated SARS-CoV-2 in the dark, being verteporfin the most effective. PDs treatment reduced viral load in infected Caco-2 cells, while not inducing cytotoxicity. Furthermore, light-independent treatment with temoporfin and verteporfin act on early stages of viral infection. Using lipid vehicles as membrane models, we characterized PDs interaction to the viral envelope. Verteporfin presented the lowest IC50 for viral inactivation and the highest partition coefficients (Kp) towards lipid bilayers. Curiously, although temoporfin and redaporfin presented similar Kps, redaporfin did not present light-independent antiviral activity, and only temoporfin and verteporfin caused lipid membrane disorder. In fact, redaporfin is located closer to the bilayer surface, while temoporfin and verteporfin are located closer to the centre. Our results suggest that viral envelope affinity, with penetration and destabilization of the lipid bilayer, seems critical to mediate PDs antiviral activity. Altogether, these findings open new avenues for the off-label application of temoporfin and verteporfin in the systemic treatment of COVID-19.

Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses.

We previously identified the bisbenzimide Hoechst 33342 (H42) as a potent multi-stage inhibitor of the prototypic poxvirus, the vaccinia virus (VACV), and several parapoxviruses. A recent report showed that novel bisbenzimide compounds similar in structure to H42 could prevent human cytomegalovirus replication. Here, we assessed whether these compounds could also serve as poxvirus inhibitors. Using virological assays, we show that these bisbenzimide compounds inhibit VACV spread, plaque formation, and the production of infectious progeny VACV with relatively low cell toxicity. Further analysis of the VACV lifecycle indicated that the effective bisbenzimide compounds had little impact on VACV early gene expression but inhibited VACV late gene expression and truncated the formation of VACV replication sites. Additionally, we found that bisbenzimide compounds, including H42, can inhibit both monkeypox and a VACV mutant resistant to the widely used anti-poxvirus drug TPOXX (Tecovirimat). Therefore, the tested bisbenzimide compounds were inhibitors of both prototypic and pandemic potential poxviruses and could be developed for use in situations where anti-poxvirus drug resistance may occur. Additionally, these data suggest that bisbenzimide compounds may serve as broad-activity antiviral compounds, targeting diverse DNA viruses such as poxviruses and betaherpesviruses.IMPORTANCEThe 2022 mpox (monkeypox) outbreak served as a stark reminder that due to the cessation of smallpox vaccination over 40 years ago, most of the human population remains susceptible to poxvirus infection. With only two antivirals approved for the treatment of smallpox infection in humans, the need for additional anti-poxvirus compounds is evident. Having shown that the bisbenzimide H33342 is a potent inhibitor of poxvirus gene expression and DNA replication, here we extend these findings to include a set of novel bisbenzimide compounds that show anti-viral activity against mpox and a drug-resistant prototype poxvirus mutant. These results suggest that further development of bisbenzimides for the treatment of pandemic potential poxviruses is warranted.

Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve throughout the coronavirus disease-19 (COVID-19) pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2, BA.2.75 and EG.5.1. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75 and EG.5.1) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.

MEK/ERK activation plays a decisive role in Zika virus morphogenesis and release.

Brazil has experienced an increase in outbreaks caused by flaviviruses. The high incidence of dengue fever, the morbidity of Zika in children, and the high mortality of yellow fever have affected millions in recent years. Deciphering host-virus interactions is important for treating viral infections, and the mitogen-activated protein kinases (MAPK) are an interesting target because of their role in flavivirus replication. In particular, mitogen-activated protein kinase kinase (MEK), which targets extracellular-signal-regulated kinase (ERK), is necessary for dengue and yellow fever infections. In this study, we evaluated the role of the MEK/ERK pathway and the effect of the MEK inhibitor trametinib on the Asian ZIKV strain PE243 and the prototype African ZIKV strain MR766, addressing genome replication, morphogenesis, and viral release. ZIKV infection stimulated ERK phosphorylation in Vero cells at 12 and 18 hours postinfection (hpi). Trametinib showed sustained antiviral activity, inhibiting both ZIKV strains for at least four days, and electron microscopy showed probable inhibition of ZIKV morphogenesis. ZIKV PE243 can complete one cycle in Vero cells in 14 hours; genome replication was detected around 8 hpi, intracellular viral particles at 12 hpi, and extracellular progeny at 14 hpi. Treatments at 6-hour intervals showed that trametinib inhibited late stages of viral replication, and the titration of intra- or extracellular virions showed that the treatment especially affected viral morphogenesis and release. Thus, ZIKV stimulated ERK phosphorylation during viral morphogenesis and release, which correlated with trametinib inhibiting both the signaling pathway and viral replication.

A study of the MAYV replication cycle: Correlation between the kinetics of viral multiplication and viral morphogenesis.

Mayaro virus (MAYV) is mainly found in Central and South America and causes a febrile illness followed by debilitating arthritis and arthralgia similar to chikungunya virus (CHIKV). Infection leads to long-term sequelae with a direct impact on the patient's productive capacity, resulting in economic losses. Mayaro fever is a neglected disease due to the limited epidemiological data. In Brazil, it is considered a potential public health risk with the number of cases increasing every year. Most of our knowledge about MAYV biology is inferred from data obtained from other alphaviruses as well as more recent studies on MAYV. Here, we analyzed the kinetics of viral replication through standard growth curves, quantification of intracellular and extracellular particles, and RNA quantification. We compared transmission electron microscopy data during different stages of infection. This approach allowed us to establish a chronological order of events during MAYV replication and its respective timepoints including cell entry through clathrin-mediated endocytosis occurring at 15-30 min, genome replication at 2-3 h, morphogenesis at 4 hpi, and release at 4-6 hpi. We also present evidence of uncharacterized events such as ribosome reorganization as well as clusters of early viral precursors and release through exocytosis in giant forms. Our work sheds new and specific light on the MAYV replication cycle and may contribute to future studies on the field.

Evaluation of the Sealing Ability and Bond Strength of Two Endodontic Root Canal Sealers: An In Vitro Study.

BACKGROUND: Obturation represents a critical step in endodontic treatment, which relies on a core material and a sealer. This study aims to evaluate the sealing ability and bond strength to the root canal walls of an epoxy resin-based sealer (AH-Plus®, Dentsply Sirona, Johnson City, TN, USA) and a bioceramic sealer (GuttaFlow Bioseal®, Coltène/Whaledent, GmbH + Co. KG, Langenau, Germany). METHODS: Thirty-eight maxillary anterior teeth with single roots and identical round sections were separated into two experimental groups according to the root canal sealers used, namely, G1 = AH-Plus® and G2 = GuttaFlow Bioseal®, and two control groups, specifically, G3 = the negative control and G4 = the positive control. The sealing capacity was measured by the penetration of the radioactive isotope 99mTc. For the push-out test, the compressive force test was performed in a universal machine and the force was applied by exerting pressure on the surface of the material to be tested in the apical to the coronal direction and using three test points with different diameters. RESULTS: GuttaFlow Bioseal® exhibited superior sealing ability compared to AH-Plus® (p = 0.003). Regarding the bond strength, AH-Plus® provided higher adhesion values than GuttaFlow Bioseal® in the three sections of the tooth root (p = 0.001). CONCLUSIONS: GuttaFlow Bioseal® had significantly better sealing ability than AH-Plus® but lower adhesion values in the three zones of the root canal, with statistically significant differences between the groups. However, it is important to note that for the action of endodontic sealers to be maximized, the root-filling technique must be most appropriate.

Do adrenergic alpha-antagonists increase the risk of poor cardiovascular outcomes? A systematic review and meta-analysis.

Due to concerns regarding neurohormonal activation and fluid retention, adrenergic alpha-1 receptor antagonists (A1Bs) are generally avoided in the setting of heart disease, namely, symptomatic heart failure (HF) with reduced ejection fraction (HFrEF). However, this contraindication is mainly supported by ancient studies, having recently been challenged by newer ones. We aim to perform a comprehensive meta-analysis aimed at ascertaining the extent to which A1Bs might influence cardiovascular (CV) outcomes. We systematically searched PubMed, Cochrane Central Register of Controlled Trials and Web of Science for both prospective and retrospective studies, published until 1 December 2020, addressing the impact of A1Bs on both clinical outcomes-namely, acute heart failure (AHF), acute coronary syndrome (ACS), CV and all-cause mortality-and on CV surrogate measures, specifically left ventricular ejection fraction (LVEF) and exercise tolerance, by means of exercise duration. Both randomized controlled trials (RCTs) and studies including only HF patients were further investigated separately. Study-specific odds ratios (ORs) and mean differences (MDs) were pooled using traditional meta-analytic techniques, under a random-effects model. A record was registered in PROSPERO database, with the code number CRD42020181804. Fifteen RCTs, three non-randomized prospective and two retrospective studies, encompassing 32 851, 19 287, and 71 600 patients, respectively, were deemed eligible; 62 256 patients were allocated to A1B, on the basis of multiple clinical indications: chronic HF itself [14 studies, with 72 558 patients, including seven studies with 850 HFrEF or HF with mildly reduced ejection fraction (HFmrEF) patients], arterial hypertension (four studies, with 44 184 patients) and low urinary tract symptoms (two studies, with 6996 patients). There were 25 998 AHF events, 1325 ACS episodes, 955 CV deaths and 33 567 all-cause deaths. When considering only RCTs, A1Bs were, indeed, found to increase AHF risk (OR 1.78, [1.46, 2.16] 95% CI, P < 0.00001, i2 2%), although displaying no significant effect on neither ACS nor CV or all-cause mortality rates (OR 1.02, [0.91, 1.15] 95% CI, i2 0%; OR 0.95, [0.47, 1.91] 95% CI, i2 17%; OR 1.1, [0.84, 1.43] 95% CI, i2 17%, respectively). Besides, when only HF patients were evaluated, A1Bs revealed themselves neutral towards not only ACS, CV, and all-cause mortality events (OR 0.49, [0.1, 2.47] 95% CI, i2 0%; OR 0.7, [0.21, 2.31] 95% CI, i2 21%; OR 1.09, [0.53, 2.23] 95% CI, i2 17%, respectively), but also AHF (OR 1.13, [0.66, 1.92] 95% CI, i2 0%). As for HFrEF and HFmrEF, A1Bs were found to exert a similarly inconsequential effect on AHF rates (OR 1.01, [0.5-2.05] 95% CI, i2 6%). Likewise, LVEF was not significantly influenced by A1Bs (MD 1.66, [-2.18, 5.50] 95% CI, i2 58%). Most strikingly, exercise tolerance was higher in those under this drug class (MD 139.16, [65.52, 212.8] 95% CI, P < 0.001, i2 26%). A1Bs do not seem to exert a negative influence on the prognosis of HF-and even of HFrEF-patients, thus contradicting currently held views. These drugs' impact on other major CV outcomes also appear trivial and they may even increment exercise tolerance.

Parainfluenza Fusion Peptide Promotes Membrane Fusion by Assembling into Oligomeric Porelike Structures.

Paramyxoviruses are enveloped viruses harboring a negative-sense RNA genome that must enter the host's cells to replicate. In the case of the parainfluenza virus, the cell entry process starts with the recognition and attachment to target receptors, followed by proteolytic cleavage of the fusion glycoprotein (F) protein, exposing the fusion peptide (FP) region. The FP is responsible for binding to the target membrane, and it is believed to play a crucial role in the fusion process, but the mechanism by which the parainfluenza FP (PIFP) promotes membrane fusion is still unclear. To elucidate this matter, we performed biophysical experimentation of the PIFP in membranes, together with coarse grain (CG) and atomistic (AA) molecular dynamics (MD) simulations. The simulation results led to the pinpointing of the most important PIFP amino acid residues for membrane fusion and show that, at high concentrations, the peptide induces the formation of a water-permeable porelike structure. This structure promotes lipid head intrusion and lipid tail protrusion, which facilitates membrane fusion. Biophysical experimental results validate these findings, showing that, depending on the peptide/lipid ratio, the PIFP can promote fusion and/or membrane leakage. Our work furthers the understanding of the PIFP-induced membrane fusion process, which might help foster development in the field of viral entry inhibition.

Targeting Zika Virus with New Brain- and Placenta-Crossing Peptide-Porphyrin Conjugates.

Viral disease outbreaks affect hundreds of millions of people worldwide and remain a serious threat to global health. The current SARS-CoV-2 pandemic and other recent geographically- confined viral outbreaks (severe acute respiratory syndrome (SARS), Ebola, dengue, zika and ever-recurring seasonal influenza), also with devastating tolls at sanitary and socio-economic levels, are sobering reminders in this respect. Among the respective pathogenic agents, Zika virus (ZIKV), transmitted by Aedes mosquito vectors and causing the eponymous fever, is particularly insidious in that infection during pregnancy results in complications such as foetal loss, preterm birth or irreversible brain abnormalities, including microcephaly. So far, there is no effective remedy for ZIKV infection, mainly due to the limited ability of antiviral drugs to cross blood-placental and/or blood-brain barriers (BPB and BBB, respectively). Despite its restricted permeability, the BBB is penetrable by a variety of molecules, mainly peptide-based, and named BBB peptide shuttles (BBBpS), able to ferry various payloads (e.g., drugs, antibodies, etc.) into the brain. Recently, we have described peptide-porphyrin conjugates (PPCs) as successful BBBpS-associated drug leads for HIV, an enveloped virus in which group ZIKV also belongs. Herein, we report on several brain-directed, low-toxicity PPCs capable of targeting ZIKV. One of the conjugates, PP-P1, crossing both BPB and BBB, has shown to be effective against ZIKV (IC50 1.08 µM) and has high serum stability (t1/2 ca. 22 h) without altering cell viability at all tested concentrations. Peptide-porphyrin conjugation stands out as a promising strategy to fill the ZIKV treatment gap.

Assessment of nanoparticle immersion depth at liquid interfaces from chemically equivalent macroscopic surfaces.

HYPOTHESIS: We test whether the wettability of nanoparticles (NPs) straddling at an air/water surface or oil/water interface can be extrapolated from sessile drop-derived macroscopic contact angles (mCAs) on planar substrates, assuming that both the nanoparticles and the macroscopic substrates are chemically equivalent and feature the same electrokinetic potential. EXPERIMENTS: Pure silica (SiO2) and amino-terminated silica (APTES-SiO2) NPs are compared to macroscopic surfaces with extremely low roughness (root mean square [RMS] roughness ≤ 2 nm) or a roughness determined by a close-packed layer of NPs (RMS roughness âˆ¼ 35 nm). Equivalence of the surface chemistry is assessed by comparing the electrokinetic potentials of the NPs via electrophoretic light scattering and of the macroscopic substrates via streaming current analysis. The wettability of the macroscopic substrates is obtained from advancing (ACAs) and receding contact angles (RCAs) and in situ synchrotron X-ray reflectivity (XRR) provided by the NP wettability at the liquid interfaces. FINDINGS: Generally, the RCA on smooth surfaces provides a good estimate of NP wetting properties. However, mCAs alone cannot predict adsorption barriers that prevent NP segregation to the interface, as is the case with the pure SiO2 nanoparticles. This strategy greatly facilitates assessing the wetting properties of NPs for applications such as emulsion formulation, flotation, or water remediation.

In-Depth Characterization of the Chikungunya Virus Replication Cycle.

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (it was first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found infection cellular activities other than those usually described for the chikungunya virus replication cycle, i.e., we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area, and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages, as follows: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

An empirical investigation on the challenges of creating custom static analysis rules for defect localization.

Custom static analysis rules, i.e., rules specific for one or more applications, have been successfully applied to perform corrective and preventive software maintenance. Pattern-driven maintenance (PDM) is a method designed to support the creation of such rules during software maintenance. However, as PDM was recently proposed, few maintainers have reported on its usage. Hence, the challenges and skills needed to apply PDM properly are unknown. In this paper, we investigate the challenges faced by maintainers on applying PDM for creating custom static analysis rules for defect localization. We conducted an observational study on novice maintainers creating custom static analysis rules by applying PDM. The study was divided into three tasks: (i) identifying a defect pattern, (ii) programming a static analysis rule to locate instances of the pattern, and (iii) verifying the located instances. We analyzed the efficiency and acceptance of maintainers on applying PDM and their comments on task challenges. We observed that previous knowledge on debugging, the subject software, and related technologies influenced the performance of maintainers as well as the time to learn the technology involved in rule programming. The results strengthen our confidence that PDM can help maintainers in producing custom static analysis rules for locating defects. However, a proper selection and training of maintainers is needed to apply PDM effectively. Also, using a higher level of abstraction can ease static analysis rule programming for novice maintainers.

Genetic diversity and population structure of Cynara cardunculus L. in southern Portugal.

Cynara cardunculus L. is a cardoon species native to the Mediterranean region, which is composed of three botanical taxa, each having distinct biological characteristics. The aim of this study was to examine wild populations of C. cardunculus established in Portugal, in order to determine their genetic diversity, geographic distribution, and population structure. Based on SSR markers, 121 individuals of C. cardunculus from 17 wild populations of the Portuguese Alentejo region were identified and analysed. Ten SSRs were found to be efficient markers in the genetic diversity analysis. The total number of alleles ranged from 9 to 17 per locus. The expected and observed means in heterozygosity, by population analysed, were 0.591 and 0.577, respectively. The wild population exhibited a high level of genetic diversity at the species level. The highest proportion of genetic variation was identified within a geographic group, while variation was lower among groups. Geographic areas having highest genetic diversity were identified in Alvito, Herdade da Abóboda, Herdade da Revilheira and Herdade de São Romão populations. Moreover, significant genetic differentiation existed between wild populations from North-Alentejo geographic locations (Arraiolos, Évora, Monte da Chaminé) and Centro Hortofrutícola, compared with other populations. This study reports genetic diversity among a representative number of wild populations and genotypes of C. cardunculus from Portugal. These results will provide valuable information towards future management of C. cardunculus germplasm.

Penetrating the Blood-Brain Barrier with New Peptide-Porphyrin Conjugates Having anti-HIV Activity.

Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide-drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide-porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.

Synergistic and Competitive Adsorption of Hydrophilic Nanoparticles and Oil-Soluble Surfactants at the Oil-Water Interface.

Fundamental insights into the interplay and self-assembly of nanoparticles and surface-active agents at the liquid-liquid interface play a pivotal role in understanding the ubiquitous colloidal systems present in our natural surroundings, including foods and aquatic life, and in the industry for emulsion stabilization, drug delivery, or enhanced oil recovery. Moreover, well-controlled model systems for mixed interfacial adsorption of nanoparticles and surfactants allow unprecedented insights into nonideal or contaminated particle-stabilized emulsions. Here, we investigate such a model system composed of hydrophilic, negatively, and positively charged silica nanoparticles and the oil-soluble cationic lipid octadecyl amine with in situ synchrotron-based X-ray reflectometry, which is analyzed and discussed jointly with dynamic interfacial tensiometry. Our results indicate that negatively charged silica nanoparticles only adsorb if the oil-water interface is covered with the positively charged lipid, indicating synergistic adsorption. Conversely, the positively charged nanoparticles readily adsorb on their own, but compete with octadecyl amine and reversibly desorb with increasing concentrations of the lipid. These results further indicate that with competitive adsorption, an electrostatic exclusion zone exists around the adsorbed particles. This prevents the adsorption of lipid molecules in this area, leading to a decreased surface excess concentration of surfactants and unexpectedly high interfacial tension.

Evaluation of kinase inhibitors as potential therapeutics for flavivirus infections.

The recent introduction of Zika virus (ZIKV), the recurrence of dengue virus (DENV), and the lethality of yellow fever virus (YFV) have had a significant impact on Brazilian society and public health. Here, we targeted two cellular kinases implicated in cell proliferation and cancer that are also important for viral replication: mitogen-activated protein kinase kinase (MEK) and Src. We used two MEK inhibitors - trametinib and selumetinib - and two Src inhibitors - saracatinib and bosutinib - to inhibit ZIKV, DENV, and YFV replication in cell culture. The cytotoxicity of the four inhibitors was determined by the observation of abnormal morphology and quantification of adherent cells by crystal violet staining. The antiviral activity of these drugs was assessed based on the reduction of plaque-forming units in cell culture as evidence of the inhibition of the replication of the selected flaviviruses. All four inhibitors showed antiviral activity, but among them, trametinib was the safest and most efficacious against all of the viruses, inhibiting the replication of ZIKV and YFV by 1000-fold, and DENV2/3 by nearly 100-fold. This pan-antiviral effect shows that trametinib could be repurposed for the treatment of flaviviral infections.

Detection of SARS-CoV-2 RNA on public surfaces in a densely populated urban area of Brazil: A potential tool for monitoring the circulation of infected patients.

The world is experiencing the worst global health crisis in recent decades since December/2019 due to a new pandemic coronavirus. The COVID-19 disease, caused by SARS-CoV-2, has resulted in more than 30 million cases and 950 thousand deaths worldwide as of September 21, 2020. Determining the extent of the virus on public surfaces is critical for understanding the potential risk of infection in these areas. In this study, we investigated the presence of SARS-CoV-2 RNA on public surfaces in a densely populated urban area in Brazil. Forty-nine of 933 samples tested positive (5.25%) for SARS-CoV-2 RNA, including samples collected from distinct material surfaces, including metal and concrete, and distinct places, mainly around hospital care units and public squares. Our data indicated the contamination of public surfaces by SARS-CoV-2, suggesting the circulation of infected patients and the risk of infection for the population. Constant monitoring of the virus in urban areas is required as a strategy to fight the pandemic and prevent further infections.

En Pointe: Dancers Report Their Pain Less Variably Than Do Controls.

The subjective nature of pain and the lack of a gold standard for objective measurement hinders effective assessment, diagnosis, and treatment. Some individuals, such as professional dancers, are better in assessing and reporting bodily sensations. This observational study aimed to assess whether dancers report their pain less variably, than other people do. After consenting, subjects completed the focused analgesia selection test (FAST), which assesses subjects' variability of pain reports. FAST outcomes, ICC and R2 reflect the magnitude of variability of pain reports observed. In addition, subjects underwent a taste task, which similarly assesses variability of tastes (salty and sweet) intensity reports and completed the Multidimensional Assessment of Interoceptive Awareness questionnaire. Thirty-three professional dancers and 33 healthy aged-matched controls were recruited. The dancers exhibited less variability of pain reports then controls (P = .013), but not in case of tastes-reports. Years of practice was positively correlated with pain reporting variability (r = .447, P = .009, and r = .380, P = .029; for FAST ICC and R2, respectively). Multidimensional Assessment of Interoceptive Awareness subscores correlated with pain reporting variability: R2 and ICC with emotional awareness (r = .260, P = .040, and r = .274, P = .030, respectively), and R2 with trusting [r = .254, P = .044]). PERSPECTIVE: The difference between dancers and controls in the magnitude of variability of pain reports is probably due to the dancers' extensive training, which focuses on attention to body signals. Our results suggest that training can improve subjective pain reports, which are essential for quality clinical care.

Structural determinants conferring unusual long life in human serum to rattlesnake-derived antimicrobial peptide Ctn[15-34].

Ctn[15-34], a downsized version of the snake venom cathelicidin-like peptide crotalicidin (Ctn), shows an unusually high lifespan (t1/2 , approximately 12 h) in human serum, which significantly adds to its promise as an antimicrobial and antitumor agent. Herein we investigate the role of Ctn[15-34] structure on serum survival. Using a set of analogs, we show that C-terminal amidation, as well as the specific layout of the Ctn[15-34] sequence-a helical N-terminal domain followed by a hydrophobic domain-is crucial for slow degradation, and any change in their arrangement results in significantly lower t1/2 . Aside from the privileged primary structure, features such as self-aggregation can be ruled out as causes for the long serum life. Instead, studies in other protease-rich fluids suggest a key role for certain human serum components. Finally, we demonstrate that Ctn[15-34] is able to induce bacterial death even after 12-hour pre-incubation in serum, in agreement with the proteolytic data. Altogether, the results shed light on the uncommon stability of Ctn[15-34] in human serum and confirm its potential as an anti-infective lead.

A spatial stream-network approach assists in managing the remnant genetic diversity of riparian forests.

Quantifying the genetic diversity of riparian trees is essential to understand their chances to survive hydroclimatic alterations and to maintain their role as foundation species modulating fluvial ecosystem processes. However, the application of suitable models that account for the specific dendritic structure of hydrographic networks is still incipient in the literature. We investigate the roles of ecological and spatial factors in driving the genetic diversity of Salix salviifolia, an Iberian endemic riparian tree, across the species latitudinal range. We applied spatial stream-network models that aptly integrate dendritic features (topology, directionality) to quantify the impacts of multiple scale factors in determining genetic diversity. Based on the drift hypothesis, we expect that genetic diversity accumulates downstream in riparian ecosystems, but life history traits (e.g. dispersal patterns) and abiotic or anthropogenic factors (e.g. drought events or hydrological alteration) might alter expected patterns. Hydrological factors explained the downstream accumulation of genetic diversity at the intermediate scale that was likely mediated by hydrochory. The models also suggested upstream gene flow within basins that likely occurred through anemophilous and entomophilous pollen and seed dispersal. Higher thermicity and summer drought were related to higher population inbreeding and individual homozygosity, respectively, suggesting that increased aridity might disrupt the connectivity and mating patterns among and within riparian populations.