S1 Site Research Articles

A Multi-Scale Approach to Model K+ Permeation Through the KcsA Channel

K+ channels allow a very efficient passage of K+ ions through the membrane while excluding Na+ ions, and these properties are essential for life. The 3D structure of the KcsA K+ channel, solved more than 20 years ago, allows to address many relevant aspects of K+ permeation and selectivity mechanisms at the molecular level. Recent crystallographic data and molecular dynamics (MD) studies suggest that no water is normally present inside the selectivity filter (SF), which can instead accommodate four adjacent K+ ions. Using a multi-scale approach, whereby information taken from a low-level simulation approach is used to feed a high-level model, we studied the mechanism of K+ permeation through KcsA channels. More specifically, we used MD to find stable ion configurations under physiological conditions. They were characterized by two adjacent K+ ions occupying the more central positions of the SF (sites S2 and S3), while the other two K+ ions could be found at the external and internal entrances to the SF. Sites S1 and S4 were instead not occupied by K+. A continuum Bikerman–Poisson–Boltzmann model that takes into account the volume of the ions and their dehydration when entering the SF fully confirmed the MD results, showing peaks of K+ occupancy at S2, S3, and the external and internal entrances, with S1 and S4 sites being virtually never occupied by K+. Inspired by the newly found ion configuration in the SF at equilibrium, we developed a simple kinetic permeation model which, fed with kinetic rate constants assessed from molecular meta-dynamics, reproduced the main permeation properties of the KcsA channel found experimentally, including sublinear current-voltage and saturating conductance-concentration relationships. This good agreement with the experimental data also implies that the ion configuration in the SF we identified at equilibrium would also be a key configuration during permeation.

Dynamics of foliar concentrations of photosynthetic pigments in woody and herbaceous plant species in the territory of an industrial city

Background. Plants growing in urban ecosystems are negatively affected by anthropogenic activities associated with environmental pollution. Plant photosynthesis is one of the processes that are particularly affected by environmental conditions, including the presence of pollutants in the atmosphere and soil. However, the dynamics of photosynthetic pigments, namely chlorophylls (Chl) and carotenoids, in plant species growing in urban ecosystems have not been sufficiently studied. The aim of this study was to analyze the effect of technogenic load on the content of pigments of photosynthesis in the cells of woody and herbaceous plants common in industrial cities, using the example of the urban ecosystem of Lviv located in the western part of Ukraine. Materials and Methods. The study was carried out at four experimental sites within the city of Lviv with different levels and types of technogenic load. Site S1 selected in the central part of the Stryiskyi park was considered as a control one. Site S2 was chosen in an area with a combined technogenic load, including road and rail traffic, and the operation of the combined heat and power plant-1 (CHPP-1). Sites S3 and S4 were subjected mainly to vehicular traffic. Leaves of six plant species were collected at sites S1–S4, including woody plants (Acer platanoides L., Aesculus hippocastanum L. and Tilia cordata Mill.) and herbaceous plants (Plantago major L., Taraxacum officinale F.H.Wigg. and Urtica dioica L). The concentration of Chl a, Chl b, the total Chl content and the concentration of carotenoids were determined spectrophotometrically. Results. The analyzed plant species growing at site S1 had the highest levels of total Chl and carotenoids compared to other sampling sites. In plant leaves collected at sites S2–S4, the total Chl content was 1.5–3.2 times lower than in the leaves collected at the site S1. The ratio of Chl a and Chl b concentrations was generally lower in plants growing at sites S2–S4 compared to plants from site S1. The concentration of carotenoids in the leaves of woody plants collected from sites S2 and S4 was 1.4–2.4 times lower compared with site S1, while in the leaves of herbaceous plants sampled at sites S2–S4, the concentration of carotenoids was 1.5–2.6 times lower than at site S1. Conclusions. The results of the study suggest that the concentration of both chlorophyll and carotenoids in the leaves of the analyzed herbaceous and woody plant species growing in the territory of an industrial city is influenced by anthropogenic impact associated with atmospheric pollution in the areas of plant growth. These indices are lower in plants growing in areas with technogenic load compared with plants growing in the green zone of the city. At the same time, the concentration of chlorophyll in the leaves of the analyzed plant species growing under urban conditions was found to be more sensitive to anthropogenic load compared to the foliar concentration of carotenoids. Therefore, the concentration of chlorophyll in plant species growing in cities can be one of the biomarkers for assessing the level of pollution caused by anthropogenic load in urban ecosystems.

Environmental Pollution Caused by Heavy Metals and Radioactivity in Two Governorates in Iraq

This study focused on knowing the levels of pollution caused by heavy metals (iron, lead) and gamma rays in the governorates of Maysan and Basra. The highest value of gamma rays was in Maysan governorate, there is a rise in these rates, according to the comparison conducted with international research, but these values do not represent a significant danger. The samples in this study are normal and free of pollution. It was found that the highest value of gamma rays in Maysan is 0.7 μSv/h at S5 and S9. The lowest value was 0.11 μSv/h in S3. As for the heavy elements, the highest value of iron was found at 0.2811 mg/l in Karama village (S2), and the lowest value was 0.2203 mg/l in S3 and the highest value for lead was 0.2632 mg/l in Kameet and the lowest value was 0.0033 In the village of Al-Huda, the highest value of gamma rays was observed at 0.8 uSv/h in S6 and S7, and the lowest value was 0.11 μSv/h in S2. The highest value for iron Fe was 0.3452 mg/l at the site S4 Al-Burjisiya Road 17, and the lowest value was 0.2232/ml for S9 Safwan Street, while pb had the highest value of 0.2817 mg/L in S7 of Zubair farms, and the lowest value was 0.0319 mm/L in S9 of Safwan Street.

Crystal structure of a family VIII β-lactamase fold hydrolase reveals the molecular mechanism for its broad substrate scope.

Family VIII esterases present similarities to class C β-lactamases, which show nucleophilic serines located at the S-X-X-K motif instead of the G-X-S-X-G or G-D-S-(L) motif shown by other carboxylesterase families. Here, we report the crystal structure of a novel family VIII (subfamily VIII. I) esterase (EH7 ; denaturing temperature, 52.6 ± 0.3 °C; pH optimum 7.0-9.0) to deepen its broad substrate range. Indeed, the analysis of the substrate specificity revealed its capacity to hydrolyse nitrocefin as a model chromogenic cephalosporin substrate (40.4 ± 11.4 units·g-1 ), and a large battery of 66 structurally different esters (up to 1730 min-1 ), including bis(2-hydroxyethyl)-terephthalate (241.7 ± 8.5 units·g-1 ) and the mycotoxin T-2 (1220 ± 52 units·g-1 ). It also showed acyltransferase activity through the synthesis of benzyl 3-oxobutanoate (40.4 ± 11.4 units·g-1 ) from benzyl alcohol and vinyl acetoacetate. Such a broad substrate scope is rare among family VIII esterases and lipolytic enzymes. Structural analyses of free and substrate-bound forms of this homooctamer esterase suggest that EH7 presents a more opened and exposed S1 site having no steric hindrance for the entrance of substrates to the active site, more flexible R1, R2 and R3 regions allowing for the binding of a wide spectrum of substrates into the active site, and small residues in the conserved motif Y-X-X containing the catalytic Tyr enabling the entrance of large substrates. These unique structural elements in combination with docking experiments allowed us to gain valuable insights into the substrate specificity of this esterase and possible others belonging to family VIII.

Impact of Hindiya Dam on the Limnological Features of Euphrates River to the North of Babil Governorate, Iraq

Five sites were chosen to the north of Babil Governorate in order to identify the limnological features and the impact of the Hindiya Dam during 2019. Site2 was located near the dam to reflect the ecological features of this site, whereas other sites, S1 was located at the upstream of the dam as a control site. Moreover, the two other sites S3 and S4 were located down the dam. The results of the study showed a close correlation between air and water temperature at all sites. Also there were significant differences in average of thirteen out of eighteen water parameters.Water temperature, total alkalinity, bicarbonate, DO, POS, TH and Mg+2 ions decreased from 22.76˚C, 203.33 mg/L, 146.75 mg/L, 8.8 mg/L, 100.023%, 438 mg/L, 35.55mg/L, respectively on S2 to 22.66 ˚C, 200.28 mg/L, 145.58 mg/L, 8.35 mg/L, 95.45%, 422.66 mg/L, 28.81 mg/L, respectively at site below the dam. While, turbidity, TSS, Ca+2, SO2+4, NO3-2 and PO4-2 increased from 13.32 NTU, 23.08 mg/L, 116.89 mg/L, 107.5 mg/L, 1.027 mg/L and 0.019 mg/L, respectively on S2 to 26.574 NTU, 26.83 mg/L, 119.23 mg/L, 137.5 mg/L, 1.145 mg/L and 0.032 mg/L, respectively at the site below the dam. However, no apparent dam effect was reported in EC, S, TDS, pH values at Euphrates River. No significant differences of all characteristics were found among sites except, turbidity, TSS, Mg+2, SO+4 and PO4-2. It can be considered that the Euphrates River is very hard, oligohaline, slightly alkaline, well ventilated and clear to turbid. In addition, the turbidity and TSS values exceeded permissible limits of water quality for aquatic life in most samples especially in site below the dam. However, the river was considered by BOD5 to be doubtful in its cleanliness water. Calcium, magnesium, nitrate and phosphate values in this study were within permissible limits.

The Relationship between Benthic Macroinvertebrate Assemblages and Water Quality Parameters in the Sanyati Basin, Lake Kariba, Zimbabwe.

Biological monitoring of reservoirs is important in assessing aquatic health. This study aimed at assessing the structure of benthic macroinvertebrate communities in relation to physicochemical parameters along Sanyati basin shoreline in Lake Kariba, Zimbabwe. Six sites (S1 to S6) characterized by various human disturbances were sampled for physicochemical parameters and benthic macroinvertebrates from January to March 2018. We computed macroinvertebrate metrics and classified them into functional feeding groups (FFGs). A canonical correspondence analysis (CCA) triplot was constructed to assess species-physicochemical relations. Significant differences across the sampling sites were observed for pH, electrical conductivity, turbidity, nitrate-nitrogen (NO3-N), ammonium nitrogen (NH4-N), total nitrogen (TN), phosphate-phosphorus (PO4-P), total phosphates (TP), and dissolved oxygen (DO). The results from CCA highlighted that S1 was generally associated with high pH, NH4-N, and TN, and Oligochaeta, Syrphidae, and Hydrophilidae families. The highest percentage of EPT taxa (39.83%) was recorded at S6, while the lowest was recorded at S1. The taxa were made up of 50% predators, 26% collector-gatherers, 6% scrappers, 6% shredders, and 3% collector-filters with 3 taxa (Chironomidae, Hydropsychidae, and Leptoceridae) having more than two FFGs. Site S1 had a significantly higher mean abundance of collector-gatherers than the other sites. A high correlation between water parameters and SASS and ASPT scores was observed indicating their ability to detect environmental changes. These findings suggest that macroinvertebrate communities are good candidates for delineating the effects of industrial pollution on water quality.

Bioaccessibility and Toxicity Assessment of Polycyclic Aromatic Hydrocarbons in Two Contaminated Sites.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous soil contaminants, and their bioaccessibility determines their environmental risks in contaminated land. In the present study, the residual concentrations of PAHs in the soils of two industrial sites were determined, and their bioaccessibility was estimated by the hydroxypropyl-β-cyclodextrin extraction (HPCD) extraction method. The results showed heavy PAH contamination at both site S1 (0.38-3342.5mgkg-1) and site S2 (0.2-138.18mgkg-1), of which high molecular weight (HMW) PAHs (4-, 5-, and 6-ring compounds) accounted for approximately 80%. The average bioaccessibility of PAHs at sites S1 and S2 was 52.02% and 29.28%, respectively. The bioaccessibility of certain PAH compounds decreased with increasing ring number of the molecule. Lower PAH bioaccessibility was detected in loamy and silty soil textures than in sandy soil. Moreover, among the soil properties, the dissolved organic matter, total organic carbon, total potassium, and total manganese concentrations had significant effects on the bioaccessibility of PAHs. The toxicity analysis showed that the composition and bioaccessibility of PAHs could affect their potential toxicity in soil. We suggest that bioaccessibility should be taken into consideration when assessing the toxicity of PAHs in soil, and more attention should be given to low-ring PAHs with high bioaccessibility.

Identification of the Stable Pt Single Sites in the Environment of Ions: From Mechanism to Design Principle.

For single-atom (SA)-based catalysis, it is urgent to understand the nature and dynamic evolution of SA active sites during the reactions. In this work, an example of Pt SA-Zn0.5 Cd0.5 S (Pt SA-ZCS) is found to display interesting phenomena when facing the Brownian collision of ions in aqueous photocatalysis. Via synchrotron radiation, surface techniques, microscopy, and theory calculations, the results show that two kinds of Pt sites exist: PtZn-sub -S3 (Pt substituting the Zn site) and Ptads -S2 (Pt adsorbing on the surface). In Na2 S, the S2- can coordinate with Pt atoms and peel them from the Ptads -S2 sites, but leaves more stable PtZn-sub -S3 sites, bringing a low but stable catalytic activity (19.40mmol g-1 h-1 ). Meanwhile, in ascorbic acid, the ascorbic acid ions show less complex ability with Pt atoms, but can decrease the migration barrier of Ptads -S2 sites (67.18 down to 35.96mmol g-1 h-1 , 52.03% drop after 6 h). Therefore, the Ptads -S2 sites gradually aggregate into nanoclusters, bringing a high but decayed catalytic activity. Moreover, a Pt SA-ZCS-Sulfur composite is designed mainly covered by PtZn-sub -S3 sites accordingly (max: 79.09mmol g-1 h-1 , 5% drop after 6 h and QE: 14.0% at 420nm), showing a beneficial strategy "from mechanism to design principle."

A Structural Comparison of SARS-CoV-2 Main Protease and Animal Coronaviral Main Protease Reveals Species-Specific Ligand Binding and Dimerization Mechanism.

Animal coronaviruses (CoVs) have been identified to be the origin of Severe Acute Respiratory Syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and probably SARS-CoV-2 that cause severe to fatal diseases in humans. Variations of zoonotic coronaviruses pose potential threats to global human beings. To overcome this problem, we focused on the main protease (Mpro), which is an evolutionary conserved viral protein among different coronaviruses. The broad-spectrum anti-coronaviral drug, GC376, was repurposed to target canine coronavirus (CCoV), which causes gastrointestinal infections in dogs. We found that GC376 can efficiently block the protease activity of CCoV Mpro and can thermodynamically stabilize its folding. The structure of CCoV Mpro in complex with GC376 was subsequently determined at 2.75 Å. GC376 reacts with the catalytic residue C144 of CCoV Mpro and forms an (R)- or (S)-configuration of hemithioacetal. A structural comparison of CCoV Mpro and other animal CoV Mpros with SARS-CoV-2 Mpro revealed three important structural determinants in a substrate-binding pocket that dictate entry and release of substrates. As compared with the conserved A141 of the S1 site and P188 of the S4 site in animal coronaviral Mpros, SARS-CoV-2 Mpro contains N142 and Q189 at equivalent positions which are considered to be more catalytically compatible. Furthermore, the conserved loop with residues 46–49 in animal coronaviral Mpros has been replaced by a stable α-helix in SARS-CoV-2 Mpro. In addition, the species-specific dimerization interface also influences the catalytic efficiency of CoV Mpros. Conclusively, the structural information of this study provides mechanistic insights into the ligand binding and dimerization of CoV Mpros among different species.

Insights Into the Na+‐Dependent Anion Exchange in the SLC4 Family From the Cryo‐EM Structure of the Sodium‐Driven Chloride/Bicarbonate Exchanger SLC4A8

SLC4 transporters are involved in the transport of HCO3‾, CO32‾, Na+, Cl‾ and H+ required for regulation of pH and ion homeostasis in several important physiological processes. Dysfunction of these transporters can lead to acid‐base abnormalities, pH dysregulation, and various disease processes. Understanding of their transport mechanism is therefore critical for development of pharmacological strategies for SLC4‐related diseases. The studies of the SLC4 transport mechanisms have been hindered by lack of information on the specific substrate binding sites within the protein matrix. No binding sites have been resolved in the two previously published structures of SLC4 proteins, the chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1) and the electrogenic sodium carbonate cotransporter 1 (NBCe1, SLC4A4), although putative binding sites have been identified on the basis of these structures from computational modelling and functional mutagenesis experiments. Here we present a new cryo‐EM structure at 3.4Å of the outward facing open (OF) conformation of the sodium dependent chloride/bicarbonate exchanger (NDCBE, SLC4A8) [1], which exchanges Na+‐CO32‾ for Cl‾, combining the feature functionalities of both AE1 and NBCe1. The structure resolves for the first time a Na+ and a CO32‾ ion bound at the center of the protein in the area of the previously identified putative binding site S1 in NBCe1 and AE1. Mutation of the residues involved in ion binding leads to significant impairment of NDCBE transport. Computational modelling with the new NDCBE structure outlines two separate ion pathways for cations and anions in the OF cavity and demonstrates that a Na+ ion bound at D800 is necessary for anion binding in site S1 and that a Na+‐CO32‾ ion pair in this site is the most likely ion load. A second anion binding site, S2, is also identified in the permeation cavity in proximity to site S1. We conclude that Na+‐dependent transport in the SLC4 family is influenced both by thermodynamic effects at the S1 binding site and by permeation effects governed by the residues lining the cation and anion permeation pathways in the OF cavity and site S2.[1] Wang W, Tsirulnikov K, Zhekova HR, et al. Cryo‐EM structure of the sodium‐driven chloride/bicarbonate exchanger NDCBE. Nat Commun. 2020;12: 5690

The transcription factor Atf1 lowers the transition barrier for nucleosome-mediated establishment of heterochromatin.

Transcription factors can exert opposite effects depending on the chromosomal context. The fission yeast transcription factor Atf1 both activates numerous genes in response to stresses and mediates heterochromatic gene silencing in the mating-type region. Investigating this context dependency, we report here that the establishment of silent heterochromatin in the mating-type region occurs at a reduced rate in the absence of Atf1 binding. Quantitative modeling accounts for the observed establishment profiles by a combinatorial recruitment of histone-modifying enzymes: locally by Atf1 at two binding sites and over the whole region by dynamically appearing heterochromatic nucleosomes, a source of which is the RNAi-dependent cenH element. In the absence of Atf1 binding, the synergy is lost, resulting in a slow rate of heterochromatin formation. The system shows how DNA-binding proteins can influence local nucleosome states and thereby potentiate long-range positive feedback on histone-modification reactions to enable heterochromatin formation over large regions in a context-dependent manner.

A cryptic K48 ubiquitin chain binding site on UCH37 is required for its role in proteasomal degradation.

Degradation by the 26 S proteasome is an intricately regulated process fine tuned by the precise nature of ubiquitin modifications attached to a protein substrate. By debranching ubiquitin chains composed of K48 linkages, the proteasome-associated ubiquitin C-terminal hydrolase UCHL5/UCH37 serves as a positive regulator of protein degradation. How UCH37 achieves specificity for K48 chains is unclear. Here, we use a combination of hydrogen-deuterium mass spectrometry, chemical crosslinking, small-angle X-ray scattering, nuclear magnetic resonance (NMR), molecular docking, and targeted mutagenesis to uncover a cryptic K48 ubiquitin (Ub) chain-specific binding site on the opposite face of UCH37 relative to the canonical S1 (cS1) ubiquitin-binding site. Biochemical assays demonstrate the K48 chain-specific binding site is required for chain debranching and proteasome-mediated degradation of proteins modified with branched chains. Using quantitative proteomics, translation shutoff experiments, and linkage-specific affinity tools, we then identify specific proteins whose degradation depends on the debranching activity of UCH37. Our findings suggest that UCH37 and potentially other DUBs could use more than one S1 site to perform different biochemical functions.

Basis for drug selectivity of plasmepsin IX and X inhibition in Plasmodium falciparum and vivax

Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P.vivax. WM4 inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease.

The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site.

Ischemia-induced cleavage of OPA1 at S1 site aggravates mitochondrial fragmentation and reperfusion injury in neurons

Neuronal mitochondrial dynamics are disturbed after ischemic stroke. Optic atrophy 1 (OPA1) and its GTPase activity are involved in maintaining mitochondrial cristae and inner membrane fusion. This study aimed to explore the role of OMA1-mediated OPA1 cleavage (S1-OPA1) in neurons exposed to cerebral ischemia and reperfusion. After oxygen-glucose deprivation (OGD) for 60 min, we found that mitochondrial fragmentation occurred successively in the axon and soma of neurons, accompanied by an increase in S1-OPA1. In addition, S1-OPA1 overexpression significantly aggravated mitochondrial damage in neurons exposed to OGD for 60 min and 24 h after OGD/R, characterized by mitochondrial fragmentation, decreased mitochondrial membrane potential, mitochondrial cristae ultrastructural damage, increased superoxide production, decreased ATP production and increased mitochondrial apoptosis, which was inhibited by the lysine 301 to alanine mutation (K301A). Furthermore, we performed neuron-specific overexpression of S1-OPA1 in the cerebral cortex around ischemia of middle cerebral artery occlusion/reperfusion (MCAO/R) mice. The results further demonstrated in vivo that S1-OPA1 exacerbated neuronal mitochondrial ultrastructural destruction and injury induced by cerebral ischemia-reperfusion, while S1-OPA1-K301 overexpression had no effect. In conclusion, ischemia induced neuronal OMA1-mediated cleavage of OPA1 at the S1 site. S1-OPA1 aggravated neuronal mitochondrial fragmentation and damage in a GTPase-dependent manner, and participated in neuronal ischemia-reperfusion injury.

The Effect of Geotechnical Factors in Road of Basrah, Southern Iraq

Most Basrah roads are conducted to a major failure in their layers and bases, which appears in the form of cracking, crushing, landing, as well as massive holes. The influence of geotechnical factors on the performance of Basrah roads has been studied. Sixteen sites were selected for sampling, which are Al-Medaina, West Qurna, Al-Hartha, Al-Dir, Shatt Al-Arab, Al-Tuwaisa, Hayy-Al-Hussein, Al-Shuaiba, Al-Maqal, Al-Gazara, Abu-Al-Khaseeb, Al-Ashar, Umm Qasser, Al-Zubair Petrochemical factory, and Al-Faw. The soil properties were studied up to 3 meters of depth at each location to find out the effect of geotechnical properties on roads. Laboratory tests were carried out on the samples, which included grain size analysis, Atterberg limits, gypsum content, total soluble salts, swelling potential, organic content, and standard penetration test. The results presented that the percentage of gypsum exceeded the allowable limit in the standard specifications at the sites (S7, S8, S13, S14, S16). Also, the percentage of total soluble salts exceeded the allowable limit in the standard specifications at the sites (S5, S8, S16). The results showed that the soils in S1, S2, S3, S4, S7, S11 have medium swelling potential, which requires treating before building roads on them. The results also showed that the percentage of organic materials exceeded the permissible limit in the standards specifications at the sites S1, S2, S3, S4, S5, S9, S10, S11, S12, S15. The results also showed that the cohesive soils in the first meter of the sites (S2, S5, S11), the second meter of the sites (S2, S7, S12) and the third meter of the sites (S5, S9, S10, S11, S12) are medium stiff soils, which requires treating before building roads on them. While the non-cohesive soils at the two sites (S8, S13) are medium dense soils, in two sites (S14, S16) are loose sandy soils in the first meter, while the second meter in sites (S8, S13, S14, S16) are medium dense soils, and the third meter at the site (S14) is medium dense soils.

Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity.

ABSTRACTThe spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. The “priming” of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2′ as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2′ cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

The Impact of Sulfate-Driven Anaerobic Oxidation of Methane on the Carbon Isotopic Composition in Marine Sediments: A Case Study from Two Sites in the Shenhu Area, Northern South China Sea

Abstract Authigenic carbonate in seep environments, as a direct byproduct of sulfate-driven anaerobic oxidation of methane (SD-AOM), is usually absent within the sediment column because of the requirement of a strict formation condition. In this case, the lack of a reliable carbon signal may impede the identification of SD-AOM and methane leakage. Here, carbon and oxygen isotopes, elemental compositions, AMS 14C dates in sediments, and porewater geochemistry were investigated from two sites (A27 and SH1) of the Shenhu area, northern South China Sea (SCS), to discuss how SD-AOM affects the carbon isotope in methane-affected marine sediments. Porewater results at both sites indicate the occurrence of methane diffusion from the sulfate-methane transition zone (SMTZ) below. The carbon isotopes of bulk-sediment carbonate and foraminifera show no distinctly negative excursion, reflecting that these signals are invalid in response to SD-AOM in the investigated sites. Then, a mass balance model is adopted to evaluate the δ13C value of authigenic carbonate (δ13CAC). Consequently, three intervals (A2 and A3 from site A27 and S2 from site SH1) are identified, featuring negative δ13CAC values, high TS/TOC ratios, and enhanced contents of authigenic carbonate, which are most likely influenced by SD-AOM. Considering the current SMTZ located at deeper layers, intervals A2 and S2 represent the locations of paleo-SMTZ, while interval A3 is thought to be influenced by the current methane diffusion. Interestingly, the δ13C values of total organic matter (δ13CTOC) show positive excursions within the paleo-SMTZs, which can be explained herein by the diagenetic modification. In the course of SD-AOM at the SMTZ, high rate of (methylotrophic) methanogenesis preferentially consumes lighter carbon atoms in organic matter, with the remainder being gradually more positive. Our results indicate that the exploration of a reliable methane-carbon response in systems lacking seep carbonates plays an important role in constraining SD-AOM and methane release.

Detection of Enteric Viruses from Wastewater and River Water in Botswana.

Waterborne diseases remain a public health concern in developing countries where many lack access to safe water. Water testing mainly uses bacterial indicators to assess water quality, which may not fully indicate the threat from other non-bacterial pathogens like enteric viruses. This study was done to ascertain and establish the viral load, the temporal and spatial distribution of rotavirus A and norovirus (GI and GII) in sewage and river water samples. A total of 45 samples of raw and treated sewage, and surface water, were collected from a sludge activated wastewater treatment plant in Gaborone, and after treatment from the Notwane River, Botswana, over a period of 9months (February 2016 to October 2016). Viruses were concentrated using polyethylene glycol/NaCl precipitation. Virus detection was performed using real-time polymerase chain reaction (RT-PCR). Rotavirus A was the most prevalent (84.4% positive samples), followed by Norovirus GI (48.9% positive samples), and Norovirus GII 46.7% positive samples). Detected viral loads went up to 104 genome copies per liter (copies/L) for all the viruses. The enteric viruses were detected in all the study sites with highest detection from site S1 (inlet). There was no significant association between physicochemical parameters and viral loads, except for pH which showed significant relationship with rotavirus and norovirus GII (p ≤ 0.05). This is the first study in Botswana to highlight the occurrence and quantification of the enteric viruses in treated and untreated wastewater, as well as surface water.

Design, synthesis and biological evaluation of novel 1, 3, 4-oxadiazole derivatives as potent neuraminidase inhibitors

Neuraminidase (NA) is an important target in the development of anti-influenza virus drugs. Compounds containing 1,3, 4-oxadiazole heterocycles have good biological activity and have been proved to have wide applications in antibacterial and antiviral drugs. In this paper, a series of novel 1, 3, 4-oxadiazole neuraminidase inhibitors (6a-6l) were designed and synthesized and their inhibitory activities of NA was tested in vitro. The results displayed that compound 6d exerts the best inhibitory activity (IC50 = 0.027 µM), which was obviously lower than that of oseltamivir carboxylate (OSC) (IC50 = 0.082 µM). Molecular docking analysis showed that the 1, 3, 4-oxadiazole heterocycle plays crucial part in compound 6d, and it can interact with the key arginine triad (Arg118, Arg292 and Arg 371) at the NA S1 site. The good efficacy of 6d may also be attributed to the extension of the substituted aniline ring to the 150-cavitiy. The theoretical and experimental results may provide reference for development of new anti-influenza drugs.