P1 Site Research Articles

Characterization of Kunitz-Domain Anticoagulation Peptides Derived from Acinetobacter baumannii Exotoxin Protein F6W77.

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system.

Design and biological evaluation of candidate drugs against zoonotic porcine deltacoronavirus (PDCoV)

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. PDCoV spillovers were recently detected in Haitian children with acute undifferentiated febrile illness, underscoring the urgent need to develop anti-PDCoV therapeutics. Coronavirus 3C-like protease (CoV 3CLpro) is essential for viral replication, and therefore provides an attractive target for drugs directed against CoV. Here, we initially evaluated the anti-PDCoV effect of Nirmatrelvir (PF-07321332), an FDA-approved anti-SARS-CoV-2 drug targeting viral 3CLpro. Regrettably, a very limited anti-PDCoV effect was achieved. By analyzing the binding modes of Nirmatrelvir with PDCoV 3CLpro and SARS-CoV-2 3CLpro, we demonstrated that the S2 pocket of 3CLpro is the primary factor underlying the differential inhibitory potency of Nirmatrelvir against different CoV 3CLpros. Based on the specific characteristics of the S2 pocket of PDCoV 3CLpro, four derivatives of Nirmatrelvir (compounds T1–T4) with substituted P2 moieties were synthesized. Compound T1, with an isobutyl at the P2 site, displayed improved anti-PDCoV activity invitro (cell infection model) and invivo (embryonated chicken egg infection model), and therefore is a potential candidate drug to combat PDCoV. Together, our results identify the substrate-binding mode and substrate specificity of PDCoV 3CLpro, providing insight into the optimization of Nirmatrelvir as an antiviral therapeutic agent against PDCoV.

Comparative Study of Water Quality Across Different Seasons in the Gregório River Watershed, São Paulo, Brazil

The goal of this study was to monitor the water quality in the Gregório river, São Carlos, São Paulo during the dry and rainy seasons using traditional methods such as physical and chemical analysis of the water and biological indicators. Six sampling points were monitored, ranging from the nearest location to the source of the river to its confluence with the Monjolinho river. Physical and chemical parameters of water (water surface temperature, dissolved oxygen, pH, and conductivity) were measured using a multiparameter probe. The distance between the points was determined according to the access to the site: from P1 to P2 (1328 m), from P2 to P3 (1278 m), from P3 to P4 (201 m), from P4 to P5 (820 m), and finally from P5 to P6 (1814 m). The biotic index used in conjunction with the environmental variables was the Biological Monitoring Working Party (BMWP), a qualitative index that utilizes benthic macroinvertebrates to classify water quality. Ceratopogonidae, Chironomidae, Chironomus, Enchytraeidae, Hirudinea, and Naididae were recorded at sites P1, P2, and P6 during both the dry and rainy seasons. This study, particularly at P6, it was observed that despite the higher concentration of dissolved oxygen compared to the other analyzed sites, which would indicate good water quality, the BMWP indicated that the location had a strongly polluted water quality, suggesting that environmental factors must be associated with biological indicators. It was concluded that the use of biological indicators along with environmental data provided a more reliable understanding of water quality.

Levels of Potentially Toxic and Essential Elements in Water and Estimation of Human Health Risks in a River Located at the Interface of Brazilian Savanna and Amazon Biomes (Tocantins River).

The Tocantins-Araguaia basin is one of South America's largest river systems, across three Brazilian states (Maranhão, Tocantins, and Pará), within the Legal Amazon region. Despite draining extensive Cerrado savanna and rainforest ecosystems, it has suffered significant degradation, notably in the past 40 years. Human activities, including agricultural expansion, deforestation, and the introduction of non-native species, have worsened the environmental damage, which is alarming since many residents and villages along the middle Tocantins River rely on it for water supply, recreation, and fishing. This study assessed the concentration of potentially toxic and essential elements in water samples from four sampling sites distributed along the middle Tocantins River. The monitoring occurred throughout 2023, involving the measurement of parameters both on-site and in the laboratory. Water quality and its health implications were evaluated using the Weighted Arithmetic Water Quality Index (WAWQI), the Water Quality Index (WQI), and the health risk assessment index. The levels of aluminum, copper, iron, magnesium, and selenium exceeded legal standards. Seasonal fluctuations indicate a complex dynamic influenced by climatic or seasonal factors, with February showing the highest values. Site P1, located in urban areas, exhibited elevated mean concentrations for conductivity, total dissolved solids (TDS), and chlorophyll, indicating the need for continuous monitoring. The nitrogen concentrations at P1 raise concerns regarding drinking water quality, which is a concern for the region's residents who use untreated river water. Despite seasonal variations in element concentrations, the overall WAWQI categorized all sections as "Excellent," and the WQI rated as "Good." Human health risk assessments detected no risks, but continuous monitoring and interventions are crucial for sustained water quality improvement.

Mutant TP53 promotes invasion of lung cancer cells by regulating desmoglein 3

PurposeTargeted therapies have markedly improved the prognosis of lung cancer patients; nevertheless, challenges persist, including limited beneficiary populations and the emergence of drug resistance. This study investigates the molecular mechanisms of mutant TP53 in lung cancer, aiming to contribute to novel strategies for targeted therapy.MethodsThe TCGA database was employed to delineate the mutational landscape of TP53 in lung cancer patients. Differential gene expression between TP53-mutant and wild-type patients was analyzed, followed by functional enrichment. DSG3 protein expression in lung cancer patients was assessed using IHC, and its impact on prognosis was analyzed in the TCGA database. The influence of TP53 on the downstream gene DSG3 was investigated using qPCR, ChIP-qPCR, and luciferase reporter gene assays. Protein enrichment in the DSG3 promoter region was examined through IP-MS, and the regulatory role of the HIF1-α/TP53 complex on DSG3 was explored using Co-IP, luciferase assays, and ChIP-qPCR. Molecular interactions between TP53 (R273H) and HIF1-α were detected through immunoprecipitation and molecular docking. The effects and mechanisms of DSG3 on lung cancer phenotypes were assessed through WB, transwell, and wound healing assays.ResultsTP53 mutations were present in 47.44% of patients, predominantly as missense mutations. DSG3 exhibited high expression in TP53-mutant lung cancer patients, and this elevated expression correlated with a poorer prognosis. TP53 interference led to a reduction in DSG3 mRNA expression, with TP53 mutant P53 enriching at the P2 site of the DSG3 promoter region, a recruitment facilitated by HIF1-α. The DBD region of TP53 (R273H) demonstrated interaction with HIF1-α. DSG3, activated through Ezrin phosphorylation, played a role in promoting invasion and metastasis.ConclusionsMutant TP53 facilitates lung cancer cell invasion by modulating desmoglein 3.Graphical abstractThe regulation of TP53 on DSG3 in lung cancer cell

Metal-contaminated sediment toxicity in a highly impacted Neotropical river: Insights from zebrafish embryo toxicity assays

The Fundão dam collapse was one of the largest mining-related disasters globally. It resulted in the release of mining tailings containing heavy metals, which contaminated the Doce River in southeastern Brazil. This study assessed the effects of acute exposure of Danio rerio embryos to sediments contaminated by mine tailings six years after the Fundão dam collapse. The study sites included P2, P3, and P4 in the upper Doce River, as well as site P1 on the Piranga River, an uncontaminated river. Sediment samples were analyzed for 10 metals/metalloid by atomic absorption spectrometry. In the assays, embryos were exposed to sediment from P1–P4 sites, and uncontaminated quartz was used as control sediment. Various biomarkers were applied to assess biological responses, and the integrated biomarker response (IBR) index was calculated for each site. Sediment samples revealed elevated levels of As, Cr, Cu, Hg, and Ni beyond Brazilian legislation limits. At 96-h exposure, embryo mortality rates exceeded 20% in P1, P2, and P3, higher than the control and P4 (p < 0.0001). Hatching rates ranged from 60 to 80% in P1, P2, and P3, lower than the control and P4 (p < 0.001). Larvae exposed to P2 sediment (closest to the Fundão dam) exhibited skeletal, physiological, and sensory malformations. Neurotoxicity was indicated by increased acetylcholinesterase activity and reduced spontaneous movements in embryos exposed to Doce River sediment. Contamination also increased metallothionein and heat shock protein 70 levels, along with changes in cell proliferation and apoptosis. Principal component analysis showed a good correlation between metals/metalloid in the sediment and larval morphometric endpoints. The IBR index highlighted suitable biomarkers for monitoring metal contamination in fish embryos. Overall, our findings suggest that sediment toxicity following the Fundão dam failure may compromise the sustainability of fish communities in the Doce River.

In vitro enhancement of Zika virus infection by preexisting West Nile virus antibodies in human plasma-derived immunoglobulins revealed after P2 binding site-specific enrichment.

Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998-2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infection in vitro. These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancement in vitro. Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operating in vivo may modulate ZIKV infection outcomes.IMPORTANCEWe investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998-2020) to neutralize or enhance Zika virus infection in vitro. West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, both in vitro and in vivo. Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

Dual pattern of cholesterol-induced decoupling of residue-residue interactions of Kir2.2

Cholesterol is a negative regulator of a variety of ion channels. We have previously shown that cholesterol suppresses Kir2.2 channels via residue-residue uncoupling on the inter-subunit interfaces within the close state of the channels (3JYC). In this study, we extend this analysis to the other known structure of Kir2.2 that is closer to the open state of Kir2.2 channels (3SPI) and provide additional analysis of the residue distances between the uncoupled residues and cholesterol binding domains in the two conformation states of the channels. We found that the general phenomenon of cholesterol binding leading to uncoupling between specific residues is conserved in both channel states but the specific pattern of the uncoupling residues is distinct between the two states and implies different mechanisms. Specifically, we found that cholesterol binding in the 3SPI state results in an uncoupling of residues in three distinct regions; the transmembrane domain, membrane-cytosolic interface, and the cytosolic domain, with the first two regions forming an envelope around PI(4,5)P2 and cholesterol binding sites and the distal region overlapping with the subunit-subunit interface characterized in our previous study of the disengaged state. We also found that this uncoupling is dependent upon the number of cholesterol molecules bound to the channel. We further generated a mutant channel Kir2.2P187V with a single point mutation in a residue proximal to the PI(4,5)P2 binding site, which is predicted to be uncoupled from other residues in its vicinity upon cholesterol binding and found that this mutation abrogates the sensitivity of Kir2.2 to cholesterol changes in the membrane. These findings suggest that cholesterol binding to this conformation state of Kir2.2 channels may destabilize the PI(4,5)P2 interactions with the channels while in the disengaged state the destabilization occurs where the subunits interact. These findings give insight into the structural mechanistic basis for the functional effects of cholesterol binding to the Kir2.2 channel.

Effects of Si/Zn Co‐Substitution on the Physicochemical Properties, In Vitro Angiogenic Potential, and Osteogenic Activity of β‐Tricalcium Phosphate

Zinc ions have shown promising osteogenic activity, while silicon ions promote angiogenesis. In this study, the effects of silicon doping, zinc doping, and their co‐doping on the physicochemical properties, as well as the in vitro angiogenic and osteogenic activities of β‐tricalcium phosphate (β‐TCP), are investigated. In the findings, it is revealed that silicon doping primarily occurs on the P1 site, leading to lattice stability disruption in β‐TCP and favoring its transformation into α‐TCP (α‐TCP). Notably, a significant portion of silicon ions is distributed on the particle surface. As a result, silicon‐doped TCP (Si–TCP) exhibits remarkable in vitro mineralization activity. Conversely, zinc doping contributes to stabilizing the β‐TCP phase, even at a sintering temperature of 1200 °C, preventing the formation of α‐TCP. Silicon/zinc‐co‐doped TCP (Si/Zn–TCP) displays comparable physicochemical properties to zinc‐doped TCP (Zn–TCP), thus demonstrating higher thermal stability than pure β‐TCP. Cell response evaluations indicate excellent biocompatibility for Si–TCP, Zn–TCP, and Si/Zn–TCP. Silicon doping significantly enhances in vitro angiogenic capability, while zinc doping notably improves osteogenic potential of β‐TCP. Furthermore, the bifunctional ion‐co‐doping approach in Si/Zn–TCP results in combined angiogenic promoting from Si–TCP and osteogenic stimulation from Zn–TCP. In these results, the considerable potential of Si/Zn–TCP is highlighted for bone defect repair applications.

Abstract 6747: Engineering M13 filamentous phages to target dendritic cells and elicit anti-tumor immunity

Abstract Filamentous phage is a safe and effective vaccine vector that can elicit robust immune responses by activating Toll-like receptor pathways. However, engineering neoantigen-displaying phages for cancer vaccines using phage display technology has been challenging and yielded inconsistent results. We developed a dendritic cell (DC)-targeting cancer vaccine platform with M13 filamentous phages using the phage display technology. The engineered phages express DC-targeting peptides (SLS) on p3 sites and spy-catcher proteins on p8 positions, allowing neoantigens and proteins of interest to be linked to the phage vector. We named our platform SCP (SLS-spy catcher phage). Imaging and western blot showed that spy-tagged proteins can be easily attached to SCP by simple mixing. Further studies demonstrated that targeting DCs can enhance antigen presentation and anti-cancer immunity. We tested SCP in MB49 and B16 mouse models and the serum, spleen and tumor tissues were isolated for further investigation of immune response upon treatment. We found that SCP effectively suppressed tumor growth by inducing systemic anti-tumor humoral and cell-mediated immunity. SCP also showed efficacy in late-stage tumor models. SCP treatment triggered particularly robust local anti-tumor response. Histological and flow cytometry analysis revealed increased infiltration of innate and adaptive immune cells, reduced PDL1 expression, and restricted neovascularization in the tumor microenvironment after intratumoral administration of SCP. Our study demonstrated that SCP can induce multifactorial modifications in the tumor microenvironment to impede tumor growth. SCP overcomes the limitations of traditional phage display technology, enabling a wide variety of peptides and proteins to be loaded onto the phage vectors. SCP is also cost-effective and scalable as phage production and purification processes are relatively mature. We propose that SCP can serve as a universal platform for cancer immunotherapy, and may offer novel treatment options for patients. Citation Format: Yee Man Betty Au-Yeung, Zheng Zeng, Jiandong Huang. Engineering M13 filamentous phages to target dendritic cells and elicit anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6747.

A combinatorial strategy for HRV 3C protease engineering to achieve the N-terminal free cleavage

Human rhinovirus 3C protease (HRV 3CP) has a high specificity against the substrate of LEVLFQ↓G at P1′ site, which plays an important role in biotechnology and academia as a fusion tag removal tool. However, a non-ignorable limitation is that an extra residue of Gly would remain at the N terminus of the recombinant target protein after cleavage with HRV 3CP, thus potentially causing protein mis-functionality or immunogenicity. Here, we developed a combinatorial strategy by integrating structure-guided library design and high-throughput screening of eYESS approach for HRV 3CP engineering to expand its P1′ specificity. Finally, a C3 variant was obtained, exhibiting a broad substrate P1′ specificity to recognize 20 different amino acids with the highest activity against LEVLFQ↓M (kcat/KM = 3.72 ± 0.04 mM−1∙s−1). Further biochemical and NGS-mediated substrate profiling analysis showed that C3 variant still kept its substrate stringency at P1 site and good residue tolerance at P2′ site, but with an expanded P1′ specificity. Structural simulation of C3 indicated a reconstructed S1′ binding pocket as well as new interactions with the substrates. Overall, our studies here prompt not only the practical applications and understanding of substrate recognition mechanisms of HRV 3CP, also provide new tools for other enzyme engineering.

Identification of Dual Inhibitors Targeting Main Protease (Mpro) and Cathepsin L as Potential Anti-SARS-CoV-2 Agents.

In this structure-activity relationship (SAR) study, we report the development of dual inhibitors with antiviral properties targeting the SARS-CoV-2 main protease (Mpro) and human cathepsin L (hCatL). The novel molecules differ in the aliphatic amino acids at the P2 site and the fluorine position on the phenyl ring at the P3 site. The identified dual inhibitors showed Ki values within 1.61 and 10.72 μM against SARS-CoV-2 Mpro; meanwhile, Ki values ranging from 0.004 to 0.701 μM toward hCatL were observed. A great interdependency between the nature of the side chain at the P2 site and the position of the fluorine atom was found. Three dual-targeting inhibitors exhibited antiviral activity in the low micromolar range with CC50 values >100 μM. Docking simulations were executed to gain a deeper understanding of the SAR profile. The findings herein collected should be taken into consideration for the future development of dual SARS-CoV-2 Mpro/hCatL inhibitors.

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

IN SILICO STUDY OF THE SYNERGISTIC INTERACTION OF 5-FLUOROURACIL AND CURCUMIN ANALOGUES AS INHIBITORS OF B-CELL LYMPHOMA 2 PROTEIN

Objective: The research objective is to explore the potential for synergistic inhibition of two curcumin analogs combined with 5-fluorouracil (5-FU) against the B-cell lymphoma 2 (BCL-2) protein.&#x0D; Methods: We investigated the synergistic inhibition of two curcumin analogs, namely, (1E,4E)-1,5-bis(4-hydroxyphenyl)penta-1,4-dien-3-one (AC01) and (1E,4E)-1,5-bis(3,4-dihydroxyphenyl)penta-1,4-dien-3-one (AC02), each combined with 5-FU by calculating their binding free energies and binding stability. An in silico investigation of the synergistic interaction of ligand pairs was conducted using the multiple ligand simultaneous docking (MLSD) technique with the AutoDock Vina package. The stability of interactions and binding free energies of each BCL-2 and curcumin analogs were examined by applying molecular dynamics techniques with the Gromacs package and MMPBSA method.&#x0D; Results: All ligand pairs had displayed strong binding affinity, as evidenced by highly negative free energy values, indicating a robust association with BCL-2. Molecular dynamics simulations were conducted over 100 ns, confirming good stability with controlled RMSD changes, suggesting that the ligand pairs had remained securely bound to the BCL-2 binding site. Additionally, RMSF analysis and energy decomposition had revealed that ligand interactions did not influence protein residue fluctuations during the simulation, and the protein-ligand complexes had maintained stability throughout the simulation. Furthermore, binding free energy calculations using the MMPBSA method had consistently shown negative values, signifying stable interactions with BCL-2 for all ligand pairs.&#x0D; Conclusion: In conclusion, our study revealed that AC01 and AC02, when combined with 5-FU, had the ability to intercalate into the P2 and P4 sites of BCL-2. This suggested that AC01 and AC02 held promise for further study as candidates for anticancer drugs, individually or in combination with 5-FU.

Room-Temperature Chemoselective Hydrogenation of Nitroarene Over Atomic Metal-Nonmetal Catalytic Pair.

Constructing atomic catalytic pair emerges as an attractive strategy to achieve better catalytic performance. Herein, an atomic Ir1─P1/NPG catalyst with asymmetric Ir─N2P1 sites that delivers superb activity and selectivity for hydrogenation of various functionalized nitrostyrene is reported. In the hydrogenation reaction of 3-nitrostyrene, Ir1─P1/NPG (NPG refers to N, P-codoped graphene) shows a turnover frequency of 1197h-1, while the reaction cannot occur over Ir1/NG (NG refers to N-doped graphene). Compared to Ir1/NG, the charge density of the Ir site in Ir1─P1/NPG is greatly elevated, which is conducive to H2 dissociation. Moreover, as revealed by density functional theory calculations and poisoning experiments, the P site in Ir1─P1/NPG is found able to bind nitrostyrene, while the neighboring Ir site provides H to reduce the nitro group in chemoselective hydrogenation of nitrostyrene. This work offers a successful example of establishing atomic catalytic pair for driving important chemical reactions, paving the way for the development of more advanced catalysts to further improve the catalytic performance.

Discovery of Hit Compounds Targeting the P4 Allosteric Site of K-RAS, Identified through Ensemble-Based Virtual Screening.

Mutants of Ras are oncogenic drivers of a large number of human tumors. Despite being recognized as an attractive target for the treatment of cancer, the high affinity for its substrate tagged the protein as undruggable for a few years. The identification of cryptic pockets on the protein surface gave the opportunity to identify molecules capable of acting as allosteric modulators. Several molecules were disclosed in recent years, with sotorasib and adagrasib already approved for clinical use. The present study makes use of computational methods to characterize eight prospective allosteric pockets (P1-P8) in K-Ras, four of which (P1-P4) were previously characterized in the literature. The present study also describes the results of a virtual screening study focused on the discovery of hit compounds, binders of the P4 site that can be considered as peptidomimetics of a fragment of the SOS αI helix, a guanine exchange factor of Ras. After a detailed description of the computational procedure followed, we disclose five hit compounds, prospective binders of the P4 allosteric site that exhibit an inhibitory capability higher than 30% in a cell proliferation assay at 50 μM.

EFFECT OF PIG FARMING EFFLUENTS ON LIMNOLOGICAL PARAMETERS AND PHYTOPLANKTON COMPOSITION

Pig farming plays an important role in the Brazilian economy, but it may trigger environmental issues, especially regarding the contamination of water resources. In this study, we evaluated the effects of the deposition of effluents from a pig farm on the phytoplankton assemblage composition and limnological parameters at different spots of the Divisa River - Fazenda São Luís, Aparecida de Minas/MG, Brazil. Over six months, we analyzed several variables at three different sampling sites on the surface of a coastal zone. We registered seventy-four phytoplankton genera, with Chlorophyceae and Cyanophyceae representing the most abundant classes, and Cryptophyceae and Cyanophyceae displaying the highest densities. The PERMANOVA analysis revealed dissimilarity between sampling sites P1 x P3 and P2 x P3, and the SIMPER analysis showed that Cryptophyceae and Cyanophyceae contributed to the observed dissimilarity. The correspondence canonical analysis (CCA) allowed the organization of sampled sites in three groups, in which the phytoplankton composition was distinguished according to trophic gradients. We concluded that pig farming effluents discharged without previous treatment into water bodies exert influences in both the phytoplankton community and the dynamics of its assemblage, by increasing total phosphorus and contributing for increased abundances of Cryptophyceae and Cyanophyceae in the last evaluated site (P3).

ENSO effects in the southern Gulf of California estimated from satellite data

This study assessed the impact of ENSO events in the seasonal and interannual variability of the sea surface temperature (SST) and phytoplankton biomass (expressed as chlorophyll-a, Chl-a levels) obtained from Moderate Resolution Imaging Spectroradiometer (MODIS) satellite products from June 2002 to December 2018, in two contrasting regions of the southern Gulf of California, the connection between the gulf and the Pacific Ocean, and the coastal region off Mazatlán. The generated dataset was compared with the El Niño–Southern Oscillation (ENSO) indices and a local wind-derived coastal upwelling index (CUI) computed in the coastal region off Mazatlán from the ERA5 monthly average wind. In this region, the results showed that the prevailing northwesterly winds reached 4 m s−1; however, southwesterly winds were observed during summer, mainly from June to August. The CUI showed values of up to 35 m3 s−1 per 100 m of coastline. The prevailing winds induce upwelling most of the year, with peaks in winter more intense during La Niña than during the El Niño event. Regarding SST and Chl-a levels, changes were observed with clear seasonal, semiannual, annual, and interannual variability, with 2.8 and 3.7 years of periodicities. In the coastal zone, seasonal values were maximum SST values (30–31 °C) during the summer and minimum values (20–22 °C) during the winter. The Chl-a levels showed an inverse trend, with minimum values (0.1–1.00 mg m−3) during summer and maximum values (>2 mg m−3) during winter. In the P1 site, in the connection between the gulf and the Pacific Ocean, the results showed during summer maximum SST values (29.5–31 °C) and minimum Chl-a (<0.2 mg m−3), while during winter minimum SST values (21–23 °C) and maximum Chl-a (0.3–2.0 mg m−3). Changes were also observed in both parameters owing to El Niño (high SST, low Chl-a levels) and La Niña (low SST, high Chl-a levels) conditions, which were confirmed by spectral and wavelet analyses. During the study period, La Niña conditions occurred during 2005/2006, 2007/2008, a multiyear event in 2010/2012, and 2017/2018, while El Niño conditions occurred during 2002/2003, 2006/2007, 2009/2010, and a multiyear event 2014/2016. Regarding their frequency, La Niña event occurred twice with a frequency corresponding to two years, and El Niño had a recurrence interval of between three and four years.

Understanding the microstructure evolution of Fe-induced diamond with DFT study: Etching and graphitization

Studying diamond graphitization is a crucial issue in ultra-precision machining because it sheds light on the reason for material removal during polishing. The polishing disc made of cast iron is usually used to process diamonds, and a high-quality surface can be obtained. However, the graphitization mechanism induced by iron (Fe) is still obscure. This paper employs the density-functional theory (DFT) method to elucidate the mechanism by which Fe induces diamond graphitization. The results demonstrated that Fe atoms at the metastable adsorption sites (P3) on the surface could change some C–C interactions between layers. Notably, multiple Fe atoms at the P3 sites could alter the region of surface graphitization and the structural dissociation direction. The surface area close to Fe atoms became stable, while the surrounding surface areas were susceptible to oblique dissociation into graphite-like structures. Moreover, we speculated that the material removal rate was related to the moving activation energy of Fe atoms. Based on the charge-transfer and microstructural evolution, the phenomenon of Fe-atom-induced diamond graphitization was attributed to varying the connections between sixfold chair rings and promoting their weakening and dissociation rather than breaking the structure of sixfold chair rings.

Modulation of Kex2p Cleavage Site for In Vitro Processing of Recombinant Proteins Produced by Saccharomyces cerevisiae.

Kex2 protease (Kex2p) is a membrane-bound serine protease responsible for the proteolytic maturation of various secretory proteins by cleaving after dibasic residues in the late Golgi network. In this study, we present an application of Kex2p as an alternative endoprotease for the in vitro processing of recombinant fusion proteins produced by the yeast Saccharomyces cerevisiae. The proteins were expressed with a fusion partner connected by a Kex2p cleavage sequence for enhanced expression and easy purification. To avoid in vivo processing of fusion proteins by Kex2p during secretion and to guarantee efficient removal of the fusion partners by in vitro Kex2p processing, P1', P2', P4, and P3 sites of Kex2p cleavage sites were elaborately manipulated. The general use of Kex2p in recombinant protein production was confirmed using several recombinant proteins.