Exploring the reciprocity between pioneer factors and development.

Development is regulated by coordinated changes in gene expression. Control of these changes in expression is largely governed by the binding of transcription factors to specific regulatory elements. However, the packaging of DNA into chromatin prevents the binding of many transcription factors. Pioneer factors overcome this barrier owing to unique properties that enable them to bind closed chromatin, promote accessibility and, in so doing, mediate binding of additional factors that activate gene expression. Because of these properties, pioneer factors act at the top of gene-regulatory networks and drive developmental transitions. Despite the ability to bind target motifs in closed chromatin, pioneer factors have cell type-specific chromatin occupancy and activity. Thus, developmental context clearly shapes pioneer-factor function. Here, we discuss this reciprocal interplay between pioneer factors and development: how pioneer factors control changes in cell fate and how cellular environment influences pioneer-factor binding and activity.

Chiffon triggers global histone H3 acetylation and expression of developmental genes in Drosophila embryos.

The histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT and ADA) that are essential for fly viability and have key roles in regulating gene expression. Here, we show that although the SAGA, ADA and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. We also identify a substantial decrease in histone acetylation in chiffon mutant embryos that exceeds that observed in Ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally driven DNA replication in the embryo. We identify a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process. This article has an associated First Person interview with the first author of the paper.

Patterns of hyperostosis during the ontogenic development of Atlantic spadefish (Broussonet, 1782) in Brazilian coast.

Marine teleost species of commercial interest are often reported with hyperostosis, an osteological condition that results in bone thickening. Various specimens of Atlantic Spadefish Chaetodipterus faber (n = 86) obtained from artisanal fishermen in Rio de Janeiro, Brazil, were radiographed and assessed to detect the occurrence of hyperostosis across four different size classes. Of the examined individuals, 58.62% displayed signs of hyperostosis, which manifested in eight skeletal regions, notably in the supraoccipital crest, cleithrum and supraneural areas. In the vertebral column, hyperostosis was more frequently observed in haemal spines than in neural spines, predominantly between the sixth and eighth caudal vertebrae. The smallest size class (<200 mm total length) showed a low frequency of hyperostosis at 7.89%. This frequency escalated for larger classes, reaching 94.12% in individuals measuring 200-300 mm in total length and was observed in all individuals exceeding 300 mm. Hyperostosis exhibited an ontogenetic development pattern, where both the occurrence frequencies and the sizes of the affected bones expanded in proportion to the fish size. This is the first description of the hyperostosis pattern of development for the species, an important commercial resource.

Prevalence and genetic diversity of avian haemosporidian parasites in islands within a mega hydroelectric dam in the Brazilian Amazon.

The Brazilian Amazon supports an extremely diverse avifauna and serves as the diversification center for avian malaria parasites in South America. Construction of hydroelectric dams can drive biodiversity loss by creating islands incapable of sustaining the bird communities found in intact forest sites. Besides anthropogenic actions, the presence of parasites can also influence the dynamics and structure of bird communities. Avian malaria (Plasmodium) and related haemosporidian parasites (Haemoproteus and Leucocytozoon) are a globally distributed group of protozoan parasites recovered from all major bird groups. However, no study to date has analyzed the presence of avian haemosporidian parasites in fragmented areas such as land bridge islands formed during artificial flooding following the construction of hydroelectric dams. The aim of this study is to assess the prevalence and molecular diversity of haemosporidians in bird communities inhabiting artificial islands in the area of the Balbina Hydroelectric Dam. The reservoir area covers 443,700 ha with 3546 islands on the left bank of the Uatumã River known to contain more than 400 bird species. We surveyed haemosporidian infections in blood samples collected from 445 understory birds, belonging to 53 species, 24 families, and 8 orders. Passeriformes represented 95.5% of all analyzed samples. We found a low overall Plasmodium prevalence (2.9%), with 13 positive samples (two Plasmodium elongatum and 11 Plasmodium sp.) belonging to eight lineages. Six of these lineages were previously recorded in the Amazon, whereas two of them are new. Hypocnemis cantator, the Guianan Warbling Antbird, represented 38.5% of all infected individuals, even though it represents only 5.6% of the sampled individuals. Since comparison with Plasmodium prevalence data prior to construction of Balbina is not possible, other studies in artificially flooded areas are imperative to test if anthropogenic flooding may disrupt vector-parasite relationships leading to low Plasmodium prevalence.

The interaction between the Dbf4 ortholog Chiffon and Gcn5 is conserved in Dipteran insect species.

Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell-cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon-A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4-dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon-B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon-B region is only present within insects. However, it was unclear how widely the interaction between Chiffon-B and Gcn5 was conserved among insect species. To examine this, we performed yeast two-hybrid assays using Chiffon-B and Gcn5 from a variety of insect species and found that Chiffon-B and Gcn5 interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon-B has features of acidic transcriptional activators such as Gal4 or VP16. We propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.

Expanded distribution of orthohantavirus Pergamino genotype: First record in Entre Rios province, Argentina.

Hantavirus Pulmonary Syndrome (HPS) is an emerging infectious disease of the Americas. Eight native rodent species have been identified as HPS virus reservoirs in Argentina. The aim of this work was to detect the orthohantavirus genotypes present in a rodent community that inhabits a zone where a fatal HPS case occurred within an endemic locality of Central Argentina. We captured 27 rodents with a trapping effort of 723 trap nights. We detected 14.3% of infected Akodon azarae with the Pergamino genotype. This result expands the known distribution of this orthohantavirus. Although the Pergamino genotype has not been associated with human cases, the information about its distribution is relevant for risk assessment against potential changes in the virus infectivity.

The Drosophila Dbf4 ortholog Chiffon forms a complex with Gcn5 that is necessary for histone acetylation and viability.

Metazoans contain two homologs of the Gcn5-binding protein Ada2, Ada2a and Ada2b, which nucleate formation of the ATAC and SAGA complexes, respectively. In Drosophila melanogaster, there are two splice isoforms of Ada2b: Ada2b-PA and Ada2b-PB. Here, we show that only the Ada2b-PB isoform is in SAGA; in contrast, Ada2b-PA associates with Gcn5, Ada3, Sgf29 and Chiffon, forming the Chiffon histone acetyltransferase (CHAT) complex. Chiffon is the Drosophila ortholog of Dbf4, which binds and activates the cell cycle kinase Cdc7 to initiate DNA replication. In flies, Chiffon and Cdc7 are required in ovary follicle cells for gene amplification, a specialized form of DNA re-replication. Although chiffon was previously reported to be dispensable for viability, here, we find that Chiffon is required for both histone acetylation and viability in flies. Surprisingly, we show that chiffon is a dicistronic gene that encodes distinct Cdc7- and CHAT-binding polypeptides. Although the Cdc7-binding domain of Chiffon is not required for viability in flies, the CHAT-binding domain is essential for viability, but is not required for gene amplification, arguing against a role in DNA replication.

Nipple-Sparing Mastectomy: Are We Providing Proper Prophylactic Antibiotic Coverage?

BACKGROUND: Infection after nipple-sparing mastectomy (NSM) and implant-based reconstruction (IBR) can be a devastating complication. The retained nipple may act as a portal or nidus for different ductal organisms, and as such, the bacteriology of surgical-site infections (SSIs) in this setting may not be adequately covered by current antibiotic recommendations. This study sought to evaluate SSI and reconstruction outcomes in relation to antibiotic choice and identify the different microbial species implicated. METHODS: A prospective database was reviewed for patients who underwent NSM with IBR from 2010 to 2019. Patient characteristics, operative details, antibiotic regimens, and subsequent treatment details were evaluated. The study analyzed SSI incidence, timing, and type of causative organisms. RESULTS: The study analyzed 571 NSMs with IBR performed for 347 patients (55% with direct implants and 45% with tissue expanders). The preoperative antibiotics consisted of cephalosporin alone for 65% of the patients, a more broad single-antibiotic use for 12% of the patients, and dual-coverage antibiotics for 20% of the patients. During a median follow-up of 1.7 years, SSI developed in 12% of the reconstructions, with 6% requiring prosthesis removal. The most common SSI organism cultured was Staphylococcal species. Neither pre- nor postoperative antibiotic choice was associated with incidence of infection, type of bacteria, or need for prosthetic explanation. CONCLUSION: For patients undergoing NSM with IBR, a more aggressive antibiotic choice is not associated with an improved SSI rate. Patient and treatment factors continue to carry the highest risk for SSI.

Extended ex vivo normothermic perfusion for preservation of vascularized composite allografts.

Ischemia and reperfusion injury remains a significant limiting factor for the successful revascularization of amputated extremities. Ex vivo normothermic perfusion is a novel approach to prolong the viability of the amputated limbs by maintaining physiologic cellular metabolism. This study aimed to evaluate the outcomes of extended ex vivo normothermic limb perfusion (EVNLP) in preserving the viability of amputated limbs for over 24 hours. A total of 10 porcine forelimbs underwent EVNLP. Limbs were perfused using an oxygenated colloid solution at 38°C containing washed RBCs. Five forelimbs (Group A) were perfused for 12 hours and the following 5 (Group B) until the vascular resistance increased. Contralateral forelimbs in each group were preserved at 4°C as a cold storage control group. Limb viability was compared between the 2 groups through assessment of muscle contractility, compartment pressure, tissue oxygen saturation, indocyanine green (ICG) angiography and thermography. EVNLP was performed for 12 hours in group A and up to 44 hours (24-44 hours) in group B. The final weight increase (-1.28 ± 8.59% vs. 7.28 ± 15.05%, P = .548) and compartment pressure (16.50 ± 8.60 vs. 24.00 ± 9.10) (P = .151) were not significantly different between the two groups. Final myoglobin and CK mean values in group A and B were: 875.0 ± 325.8 ng/mL (A) versus 1133.8 ± 537.7 ng/mL (B) (P = .056) and 53 344.0 ± 16 603.0 U/L versus 64 333.3 ± 32 481.8 U/L (P = .286). Tissue oxygen saturation was stable until the end in both groups. Infra-red thermography and ICG-angiography detected variations of peripheral limb perfusion. Our results suggest that extended normothermic preservation of amputated limbs is feasible and that the outcomes of prolonged EVNLP (>24 hours) are not significantly different from short EVNLP (12 hours).

Factors Influencing the Aesthetic Outcome and Quality of Life After Breast Reconstruction: A Cross-sectional Study.

BACKGROUND: The most important purpose of reconstruction is to increase or restore the patient's quality of life (QOL). The purpose of our study was to evaluate the QOL and aesthetic outcomes of patients after autologous versus implant-based breast reconstruction. METHODS: Patients who underwent breast reconstruction between 2009 and 2011 were included. The Breast-Q, a validated breast reconstruction QOL questionnaire, was used along with postoperative photographs panel analyses using a multiparameter breast-specific aesthetic outcome scale and retrospective evaluation of demographic and treatment data. RESULTS: Of 820 patients, 261 complete questionnaires were evaluated. On the multivariable linear regression, the "satisfaction with breasts" was positively influenced by autologous and bilateral reconstructions, whereas radiation therapy (RTx), the time between the reconstruction and the questionnaire, and the number of surgeries due to complications were negative factors (adjusted R = 0.183; P < 0.001). The same factors influenced the "satisfaction with the outcomes." The mean "overall breast appearance" was also positively influenced by autologous and bilateral reconstructions, and RTx and the total number of surgeries were negative predictive factors (adjusted R = 0.311, P < 0.001). CONCLUSIONS: The aesthetic result and QOL after breast reconstruction for breast cancer treatment are positively influenced by the use of autologous tissue and bilaterality. Factors that negatively influenced the aesthetic result and the QOL include use of RTx, a higher number of surgeries needed for the reconstruction, reoperations due to complications, higher body mass index, and a longer time elapsed between reconstruction and the questionnaire.

Facelift Part I: History, Anatomy, and Clinical Assessment.

In this article, the authors aim to thoroughly describe the critical surgical anatomy of the facial layers, the retaining ligamentous attachments of the face, and the complex three-dimensional course of the pertinent nerves. This is supplemented with clarifying anatomic dissections and artwork figures whenever possible to enable easy, sound, and safe navigation during surgery. The historic milestones that led the evolution of cervicofacial rejuvenation to the art we know today are summarized at the beginning, and the pearls of the relevant facial analysis that permit accurate clinical judgment and hence individualized treatment strategies are highlighted at the end. The facelift operation remains the cornerstone of face and neck rejuvenation. Despite the emergence of numerous less invasive modalities, surgery continues to be the most powerful and more durable technique to modify facial appearance. All other procedures designed to ameliorate facial aging are either built around or serve as adjuncts to this formidable craft.

Identification of potent bovine viral diarrhea virus inhibitors by a structure-based virtual screening approach.

Bovine viral diarrhea virus (BVDV) is a pestivirus whose infection in cattle is globally distributed. The use of antivirals could complement vaccination as a tool of control and reduce economic losses. The RNA-dependent RNA polymerase (RdRp) of the virus is essential for its genome replication and constitutes an attractive target for the identification of antivirals. With the aim of obtaining selective BVDV inhibitors, the crystal structure of BVDV RdRp was used to perform a virtual screening. Approximately 15,000 small molecules from commercial and in-house databases were evaluated and several structurally different compounds were tested in vitro for antiviral activity. Interestingly, of twelve evaluated compounds, five were active and displayed EC50 values in the sub and low-micromolar range. Time of drug addition experiment and measured intracellular BVDV RNA showed that compound 7 act during RNA synthesis. Molecular Dynamics and MM/PBSA calculation were done to characterize the interaction of the most active compounds with RdRp, which will allow future ligand optimization. These studies highlight the use of in silico screening to identify a new class of BVDV inhibitors.

3-Hydroxyphenylboronic Acid-Based Carbon Dot Sensors for Fructose Sensing.

The selective fluorescence sensing of fructose was achieved by fluorescence quenching of the emission of hydrothermal-synthesized carbon quantum dots prepared by 3-hydroxyphenylboronic acid. Quantification of fructose was possible in aqueous solutions with pH of 9 (Limit of Detection LOD and Limit of Quantification LOQ of 2.04 and 6.12 mM), by quenching of the emission at 376 nm and excitation ~380 nm with a linearity range of 0-150 mM. A Stern-Volmer constant (KSV) of 2.11 × 10-2 mM-1 was obtained, while a fluorescent quantum yield of 31% was calculated. The sensitivity of this assay towards fructose was confirmed by comparison with other sugars (such as glucose, sucrose and lactose). Finally, the validity of the proposed assays was further demonstrated by performing recovery assays in different matrixes. Graphical Abstract.

Recovery of lanthanides from hydrocarbon cracking spent catalyst through chemical and biotechnological strategies.

The aim of this work is to evaluate the rare earth elements (REEs) recovery from fluid catalytic cracking spent catalyst (FCC-SC) by chemical and biochemical strategies while also examining a route for the valorization of biodiesel-derived glycerin (RG), which is presently unprofitable to refine. Recovery tests for REEs were performed with no pretreatment of the FCC-SC. A chemical leaching investigation was carried out using HCl, HNO3, NaOH, CaCl2 and citric acid aqueous solutions (1 mol L-1, at 30, 50, 60 or 70 ± 1 °C). The leaching tests carried out with 1 mol L-1 citric acid at 50 °C provided the best recovery of La (27%). Subsequent bioleaching tests were carried out with four strains of Yarrowia lipolytica to evaluate their potential to produce organic acids using RG as the main carbon source. The FCC-SC contains some REEs, predominantly La. Remarkable biorecovery rates for REEs (namely, La (53%), Ce and Nd (both 99%)) were achieved using the Y. lipolytica IM-UFRJ 50678 fermented medium at 50 °C. Thus, here, a sustainable approach to recovering metals from spent cracking catalyst using RG under low-cost and non-energy-intensive processing conditions is reported.

Developing a protocol for normothermic ex-situ limb perfusion.

BACKGROUND: Ischemia time represents a significant limitation for successful extremity transplantation because of the rapid deterioration of ischemic muscle. Normothermic ex-situ preservation is an emergent method to prolong the organ viability following procurement, by replicating the physiologic conditions. The aim of this study was to develop an ex-situ normothermic limb perfusion system to preserve the viability and function of porcine limbs for 12 hours following procurement. METHODS: A total of 18 swine limbs were perfused. Thirteen limbs were used to develop the perfusion protocol. Five limbs were perfused according to the optimized protocol. These limbs were perfused at 39°C for twelve hours using an oxygenated colloid solution containing red blood cells. Glucose and electrolytes were kept within physiologic range by partial perfusate exchange. Limb specific perfusion quality was assessed by muscle contractility upon electrical nerve stimulation, compartment pressure, creatine kinase (CK) and myoglobin concentrations, tissue oxygen saturation (near infrared spectroscopy), indocyanine green angiography, and infrared radiation by thermographic imaging. RESULTS: The last five limbs reached the 12 hours' perfusion target maintaining normal compartment pressure (16.4 ± 8.20 mmHg), minimal weight increase (0.54 ± 7.35%), and mean muscle temperature of 33.6 ± 1.67°C. Myoglobin and CK concentrations were 875 ± 291.4 ng/mL, and 53344 ± 14850.34 U/L, respectively, at the end of perfusion. Muscle contraction was present in all limbs until cessation of perfusion. Differences in uniformity and quality of distal perfusion were identified with thermography and angiography imaging at 12 hours of perfusion. CONCLUSIONS: Ex-situ normothermic limb perfusion preserves swine limb physiology and function for at least 12 hours.

Biosurfactant-assisted phytoremediation of multi-contaminated industrial soil using sunflower (Helianthus annuus L.).

This study evaluated the use of commercial rhamnolipid biosurfactant supplementation in the phytoremediation of a soil via sunflower (Helianthus annuus L.) cultivation. The soil, obtained from an industrial area, was co-contaminated with heavy metals and petroleum hydrocarbons. The remediation tests were monitored for 90 days. The best results for removal of contaminants were obtained from the tests in which the sunflower plants were cultivated in soil with 4 mg kg-1 of the rhamnolipid. Under these conditions, reductions of 58% and 48% were obtained in the total petroleum hydrocarbon (TPH) and polycyclic aromatic hydrocarbon (PAH) concentrations, respectively; reductions in the concentrations of the following metals were also achieved: Ni (41%), Cr (30%), Pb (29%), and Zn (20%). The PCR-DGGE analysis of soil samples collected before and after the treatments verified that the plant cultivation and biosurfactants supplementation had little effect on the structure of the dominant bacterial community in the soil. The results indicated that sunflower cultivation with the addition of a biosurfactant is a viable and efficient technology to treat soils co-contaminated with heavy metals and petroleum hydrocarbons.

Grab and passive sampling applied to pesticide analysis in the São Lourenço river headwater in Campo Verde - MT, Brazil.

In this study, the quality of surface water in the headwaters of São Lourenço River in Mato Grosso, Brazil, was evaluated in relation to contamination by pesticides. For this purpose, samples were collected between December 2015 and June 2016 by grab sampling and by passive sampling using an integrative polar organic compound sampler installed in the field during four 14-day cycles between March and June 2016. The analyses were performed by gas chromatography (CG/MS) and by liquid chromatography (UPLC-MS/MS). The results showed the detection of two pesticides (atrazine and pyraclostrobin) of the five analyzed by passive sampling and eight active principles among the 20 analyzed (malathion, diuron, carbofuran, carbendazim, trifluralin, imidacloprid, metolachlor, and acetamiprid) by grab sampling. The detection of 10 pesticides, even almost a decade after the beginning of a recovery process of the ciliary forest, confirms the headwaters' vulnerability to these contaminants and passive sampling proved to be an important tool in capturing small concentrations of pesticides constituting an interesting complement to grab sampling.

Apparent Age is a Reliable Assessment Tool in 20 Facelift Patients.

BACKGROUND: Although the literature is replete with favorable facelift results, there are few validated facial rejuvenation outcome measures. Apparent age (AA), a visual estimate of age by objective observers, has been utilized in several studies; although attractive, AA lacks validation. OBJECTIVE: The aim of this study is to examine the reliability of AA, highlighting the importance of the exclusive use of validated outcome measures in future studies. METHODS: Ten blinded reviewers assessed pre- and postoperative photographs of 32 patients who underwent facelift. Each reviewer completed 3 surveys at 3-month intervals composed of 40 randomly ordered photos; totaling 1200 photographs assigned an AA. The intra-class correlation coefficient was classified as "excellent," "good," "fair," or "poor." The accuracy of assigned AA, agreement within 5 years, and reduction in AA were also evaluated. RESULTS: The mean difference of preoperative true age from assigned AA was 2.74 ± 4.36 years. Forty-three percent of raters were within 5-years (±2.5) of the mean. Intra-rater reliability preoperatively and postoperatively were 0.77 (95% CI, 0.82-0.72) and 0.75 (95% CI, 0.79-0.71), respectively. Inter-rater reliability preoperatively was 0.98 (95% CI, 0.99-0.96), while postoperatively was 0.95 (95% CI, 0.99-0.95). Mean AA reduction was 5.23 ± 2.81, with an intra-rater reliability 0.15 (95% CI, 0.03-0.34) and inter-rater reliability 0.65 (95% CI, 0.84-0.38). CONCLUSION: Using current statistical measures and analysis, AA is an acceptable tool for pre- and postoperative facial evaluation when assessed by a group of 10 reviewers. Therefore, apparent age represents a reliable and valid objective observer assigned measure for evaluation of facelift outcomes.

Estimating synchronous demographic changes across populations using hABC and its application for a herpetological community from northeastern Brazil.

Many studies propose that Quaternary climatic cycles contracted and/or expanded the ranges of species and biomes. Strong expansion-contraction dynamics of biomes presume concerted demographic changes of associated fauna. The analysis of temporal concordance of demographic changes can be used to test the influence of Quaternary climate on diversification processes. Hierarchical approximate Bayesian computation (hABC) is a powerful and flexible approach that models genetic data from multiple species, and can be used to estimate the temporal concordance of demographic processes. Using available single-locus data, we can now perform large-scale analyses, both in terms of number of species and geographic scope. Here, we first compared the power of four alternative hABC models for a collection of single-locus data. We found that the model incorporating an a priori hypothesis about the timing of simultaneous demographic change had the best performance. Second, we applied the hABC models to a data set of seven squamate and four amphibian species occurring in the Seasonally Dry Tropical Forests (Caatinga) in northeastern Brazil, which, according to paleoclimatic evidence, experienced an increase in aridity during the Pleistocene. If this increase was important for the diversification of associated xeric-adapted species, simultaneous population expansions should be evident at the community level. We found a strong signal of synchronous population expansion in the Late Pleistocene, supporting the increase of the Caatinga during this time. This expansion likely enhanced the formation of communities adapted to high aridity and seasonality and caused regional extirpation of taxa adapted to wet forest.

Synthesis, antiviral evaluation and molecular docking studies of N4-aryl substituted/unsubstituted thiosemicarbazones derived from 1-indanones as potent anti-bovine viral diarrhea virus agents.

A series of N4-arylsubstituted thiosemicarbazones derived from 1-indanones and a set of compounds lacking such substitution in the N4 position of the thiosemicarbazone moiety were synthesized and evaluated for their anti-bovine viral diarrhea virus (BVDV) activity. Among these, derivatives 2 and 15 displayed high activity (EC50=2.7±0.4 and 0.7±0.1µM, respectively) as inhibitors of BVDV replication. Novel key structural features related to the anti-BVDV activity were identified by structure-activity relationship (SAR) analysis. In a previous study, the thiosemicarbazone of 5,6-dimethoxy-1-indanone (5,6-TSC) was characterized as a non-nucleoside inhibitor (NNI) of the BVDV RNA-dependent RNA polymerase. In the present work, cross-resistance assays were performed with the most active compounds. Such studies were carried out on 5,6-TSC resistant BVDV (BVDV-TSCr T1) carrying mutations in the viral polymerase. This BVDV mutant was also resistant to compound 15. Molecular docking studies and MM/PBSA calculations were performed to assess the most active derivatives at the 5,6-TSC viral polymerase binding site. The differences in the interaction pattern and the binding affinity of derivative 15 either to the wild type or BVDV-TSCr T1 polymerase were key factors to define the mode of action of this compound.