Unique Site Research Articles

Proteomic Analysis of Lysine Acetylation and Succinylation to Investigate the Pathogenicity of Virulent Pseudomonas syringae pv. tomato DC3000 and Avirulent Line Pseudomonas syringae pv. tomato DC3000 avrRpm1 on Arabidopsis thaliana.

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is able to infect many economically important crops and thus causes substantial losses in the global agricultural economy. Pst DC3000 can be divided into virulent lines and avirulent lines. For instance, the pathogen effector avrRPM1 of avirulent line Pst-avrRpm1 (Pst DC3000 avrRpm1) can be recognized and detoxified by the plant. To further compare the pathogenicity mechanisms of virulent and avirulent Pst DC3000, a comprehensive analysis of the acetylome and succinylome in Arabidopsis thaliana was conducted following infection with virulent line Pst DC3000 and avirulent line Pst-avrRpm1. In this study, a total of 1625 acetylated proteins encompassing 3423 distinct acetylation sites were successfully identified. Additionally, 229 succinylated proteins with 527 unique succinylation sites were detected. A comparison of these modification profiles between plants infected with Pst DC3000 and Pst-avrRpm1 revealed significant differences. Specifically, modification sites demonstrated inconsistencies, with a variance of up to 10% compared to the control group. Moreover, lysine acetylation (Kac) and lysine succinylation (Ksu) displayed distinct preferences in their modification patterns. Lysine acetylation is observed to exhibit a tendency towards up-regulation in Arabidopsis infected with Pst-avrRpm1. Conversely, the disparity in the number of Ksu up-regulated and down-regulated sites was not as pronounced. Motif enrichment analysis disclosed that acetylation modification sequences are relatively conserved, and regions rich in polar acidic/basic and non-polar hydrophobic amino acids are hotspots for acetylation modifications. Functional enrichment analysis indicated that the differentially modified proteins are primarily enriched in the photosynthesis pathway, particularly in relation to light-capturing proteins. In conclusion, this study provides an insightful profile of the lysine acetylome and succinylome in A. thaliana infected with virulent and avirulent lines of Pst DC3000. Our findings revealed the potential impact of these post-translational modifications (PTMs) on the physiological functions of the host plant during pathogen infection. This study offers valuable insights into the complex interactions between plant pathogens and their hosts, laying the groundwork for future research on disease resistance and pathogenesis mechanisms.

Oxygen Coordination Promotes Single-Atom Cu(II)-Catalyzed Azide-Alkyne Click Chemistry without Reducing Agents.

Unique active sites make single-atom (SA) catalysts promising to overcome obstacles in homogeneous catalysis but challenging due to their fixed coordination environment. Click chemistry is restricted by the low activity of more available Cu(II) catalysts without reducing agents. Herein, we develop efficient, O-coordinated SA Cu(II) directly catalyzed click chemistry. As revealed by theoretical calculations of the superior coordination structure to promote the click reaction, an organic molecule-assisted strategy is applied to prepare the corresponding SA Cu catalysts with respective O and N coordination. Although they both belong to Cu(II) centers, the O-coordinated one exhibits a 5-fold higher activity than the other and even much better activity than traditional homogeneous and heterogeneous Cu(II) catalysts. Control experiments further proved that the O-coordinated SA Cu(II) catalyst tends to be reduced by alkyne into Cu acetylide rather than the N-coordinated catalyst and thus facilitates click chemistry.

Methane seeps on the U.S. Atlantic margin: An updated inventory and interpretative framework

Since the discovery of >570 methane flares on the northern U.S. Atlantic margin between Cape Hatteras and Georges Bank in the last decade, the acquisition of thousands of kilometers of additional water column imaging data has provided greater coverage at water depths between the outer continental shelf and the lower continental slope. The additional high-resolution data reveal >1400 gas flares, but the removal of probable duplicates from the combined database of new flares and those recognized in 2014 yields ∼1139 unique sites. Most of these sites occur in clusters of 5 or more seeps, leaving about 275 unique locations (including 47 clusters) for seepage along the margin. As a function of depth, seep distribution is heavily skewed toward the upper continental slope at water depths shallower than 400 m on the southern New England margin and ∼ 550 m in the Mid-Atlantic Bight, with additional seeps clustered at ∼1100 m and just deeper than ∼1400 m in both sectors. Despite little ongoing tectonic deformation or active faulting on this passive margin, a variety of processes driven from below the seafloor (e.g., migration of fluids along faults or through permeable strata, seepage above diapirs or other pre-existing structures) and from above (e.g., erosion, sapping, unroofing) contribute to the development of seeps in different settings along the margin. In addition, the prevalence of seeps on promontories overlooking shelf-breaking canyons may be directly related to the three-dimensional nature of the hydrate stability zone in these locations. As a function of depth, the parts of the slope at the contemporary landward limit of gas hydrate stability are devoid of seeps, and the upper slope zones with the most concentrated seepage were not within the gas hydrate stability zone even during the Last Glacial Maximum. Thus, if the large number of upper slope seeps is at least partially sourced in gas hydrate degradation, the gas emitted at these seeps must have migrated there from greater depths on the continental slope.

Identification of antibiotic-resistance markers of Edwardsiella tarda using aptamers

Edwardsiella tarda poses significant challenges in the aquaculture sector due to its antibiotic resistance, leading to substantial economic loss in the aquaculture industry. Therefore, it is imperative to identify antibiotic-resistance markers to prevent and control E. tarda. In the present study, we used tetracycline-sensitive (S) and resistant (R) E. tarda strains and six non-target bacterial strains, to develop aptamers through the Systematic Evolution of Ligands by Exponential Enrichment technique. We then conducted comparative analysis of these aptamers based on their relative importance index values and selected six candidate sequences (M1, M13, M15, N57, N69, and N71) for affinity verification. The results showed that M1 had significantly higher affinity towards both the S and R strains compared to the non-target strains. M13 and M15 had significantly higher affinities for the S strain compared to the R strain and non-target strains, and N57, N69, and N71 had significantly higher affinities for R strains relative to the sensitive and non-target strains. Subsequent analysis indicated that N57 and N69 specifically recognized unique sites in the R strain. Therefore, our study presents a novel approach for detecting and controlling antibiotic-resistant strains of E. tarda and identification of drug-resistant markers in bacteria.

A macrophage-cell model of HIV latency reveals the unusual importance of the bromodomain axis

BackgroundAlthough macrophages are now recognized as an essential part of the HIV latent reservoir, whether and how viral latency is established and reactivated in these cell types is poorly understood. To understand the fundamental mechanisms of viral latency in macrophages, there is an urgent need to develop latency models amenable to genetic manipulations and screening for appropriate latency-reversing agents (LRAs). Given that differentiated THP-1 cells resemble monocyte-derived macrophages in HIV replication mechanisms, we set out to establish a macrophage cell model for HIV latency using THP-1 cells.MethodsWe created single-cell clones of THP-1 cells infected with a single copy of the dual-labeled HIVGKO in which a codon switched eGFP (csGFP) is under the control of the HIV-1 5’ LTR promoter, and a monomeric Kusabira orange 2 (mKO2) under the control of cellular elongation factor one alpha promoter (EF1α). Latently infected cells are csGFP−, mKO2+, while cells with actively replicating HIV (or reactivated virus) are csGFP+,mKO2+. After sorting for latently infected cells, each of the THP-1 clones with unique integration sites for HIV was differentiated into macrophage-like cells with phorbol 12-myristate 13-acetate (PMA) and treated with established LRAs to stimulate HIV reactivation. Monocyte-derived macrophages (MDMs) harboring single copies of HIVGKO were used to confirm our findings.ResultsWe obtained clones of THP-1 cells with latently infected HIV with unique integration sites. When the differentiated THP-1 or primary MDMs cells were treated with various LRAs, the bromodomain inhibitors JQ1 and I-BET151 were the most potent compounds. Knockdown of BRD4, the target of JQ1, resulted in increased reactivation, thus confirming the pharmacological effect. The DYRK1A inhibitor Harmine and lipopolysaccharide (LPS) also showed significant reactivation across all three MDM donors. Remarkably, LRAs like PMA/ionomycin, bryostatin-1, and histone deacetylase inhibitors known to potently reactivate latent HIV in CD4 + T cells showed little activity in macrophages.ConclusionsOur results indicate that this model could be used to screen for appropriate LRAs for macrophages and show that HIV latency and reactivation mechanisms in macrophages may be distinct from those of CD4 + T cells.

Maackia amurensis seed lectin (MASL) and soluble human podoplanin (shPDPN) sequence analysis and effects on human oral squamous cell carcinoma (OSCC) cell migration and viability

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent.

One-step synthesis of hydroxyl-functionalized ionic hyper-cross-linked polymers with high surface areas for efficient CO2 capture and fixation

Ionic liquid-based functional materials have attracted significant attention for their distinctive structure in the field of CO2 capture and conversion. In this work, a series of hydroxyl-functionalized ionic hyper-cross-linked polymers are prepared through a one-step Friedel-Crafts reaction involving hypoxanthine (HX) and benzimidazole (BI) as the monomers, along with various halohydrocarbon crosslinking agents. These polymers demonstrate a high specific surface area (558–1480 m2·g−1), well-developed microporous structure, and unique ion sites, enabling them to exhibit remarkable and reversible CO2 adsorption properties. Particularly noteworthy is their CO2 adsorption capacity, which surpasses that of similar ionic polymers documented in the literature, reaching 157.5 mg·g−1 at 273 K and 1 bar. Additionally, these polymers function as recyclable catalysts in the cycloaddition reaction of CO2 and epoxides, enabling the conversion of CO2 into cyclic carbonates with yields of up to 99 % even without a co-catalyst. Mechanism investigation reveals that the introduction of hydroxyl groups in the polymer is the key to improving catalytic activity through a synergistic catalytic effect. This research provides a novel concept for designing ionic functional materials with capabilities in both CO2 adsorption and catalytic activity.

Development of adventure tourism in the Republic of Sakha (Yakutia)

The article examines the trends in the development of adventure tourism in the Republic of Sakha (Yakutia), where nature-oriented forms of tourism are the most promising. The object of the study is adventure tourism, the subject is the peculiarities of its development in the regions of new development on the example of the Republic of Sakha (Yakutia). The concept and characteristics of adventure tourism are briefly analysed. It is determined that adventure tourism is one of the types of nature–oriented tourism, including visits to unique natural sites (mainly in areas untouched by economic activity) through active forms of movement combined with moderate physical exertion, overcoming difficulties and obtaining unforgettable experiences, impressions and new knowledge about the world around. The specifics of the organization and trends in the development of tourism in the Republic of Sakha (Yakutia) are analysed. The analysis of the natural and recreational potential showed high attractive properties of the studied territory and the possibility of developing various types of active tourism. Despite the rich natural resource potential, the development of adventure tourism in the region is hampered by a number of negative factors. The main problems of the development of adventure tourism are considered and measures to intensify the development of adventure tourism in the region are proposed.

Abstract 6279: Estrogen signaling in low-grade serous ovarian carcinoma

Abstract High level of estrogen in the blood of women has been linked to the progression and possibly development of ovarian cancer. Almost all low-grade serous ovarian cancer (LGSOC) expresses estrogen receptor alpha (ERα protein encoded by the gene ESR1) and develops during reproductive age of women. However, recent studies suggested that a higher expression of ERα correlates with better survival. In a phase II clinical trial, hormonal therapies have been shown to be associated with a clinical benefit rate of 61% for recurrent ER-positive LGSOC patients. The study also indicated that women with recurrent LGSOC who received hormonal therapies had a significantly longer progression-free survival but not overall survival. Endocrine therapies have proven to be effective in treating breast cancer. Unfortunately, in ovarian cancer clinical trials (GOG0281 and PARAGON), these treatments have not achieved comparable results, with an objective response rate of only approximately 14%. To understand the ESR1 regulation in LGSOC, we have performed RNAseq and ChIPseq analyses of LGSOC tumor tissues. We also forced over-expression of ESR1 in LGSOC cell lines. Using RNAseq data, we aligned the sequencing reads to all the known ESR1 transcript variants. We found that ESR1 transcript variant 1, 2, 3 and X11 were the major transcripts expressed. Variants 1, 2 and 3 encode the regular 66 kDa ESR1 protein, while the X11 variant encodes a 52 kDa ESR1 protein. Both 66 kDa and 52 kDa ESR1 proteins were also detected in LGSOC tumor tissues by western blots. To further explore the function of ESR1 66kDa in LGSOC, we over-expressed ESR1 variant 1 in two LGSOC cell lines (HOC-7 and HCC5075) by transfecting with an expression plasmid. Total proteins were extracted from the mock control and transfected cells for functional proteomic analysis by reverse phase protein arrays (RPPA). From the RPPA data, overexpressing ESR1 in LGSOC cell lines suppressed DNA repair pathways but activated cell cycle and mitosis pathways. To further delineate the ESR1 DNA binding sites in LGSOC, we performed ChIPseq analysis of these tumor samples as well as two ovarian cancer cell lines that express ESR1 protein. Our results demonstrated that ESR1 regulated “cistromes” in ER positive ovarian carcinoma is very different from that in ER positive breast cancer as much less ESR1 binding sites were found in LGSOC. There are ESR1 binding sites common to both types of cancers, but we found quite a few unique ESR1 binding sites. We are trying to integrate the ChIPseq and RNAseq data and hopefully identify, for the first time, an ovarian cancer specific ERα cistrome. ER expression may not always correlate with activation of ESR1 signaling pathways, which may be one of the reasons underlying endocrine therapy resistance in ovarian cancer. The results of an integrated analyses will also give us insight on the management of ER positive LGSOC by identifying new genetic targets that may be used in combination with endocrine therapy. Citation Format: Yvonne TM Tsang, Chun Wai Oscar Ng, David Gershenson, K-Kwok wong. Estrogen signaling in low-grade serous ovarian carcinoma [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 6279.

Abstract 3320: In vivo and in vitro experience with novel direct Pan-RAS inhibitors

Abstract RAS oncogenes are frequently activated by mutations in human cancer, where they act as driving elements of the disease. Mutation rates can range from ~25% of Non Small Cell Lung cancers to over 90% of pancreatic cancers. RAS proteins can also be hyper-activated to drive cancer at a significant frequency in the absence of RAS gene mutations when their protein regulatory systems are damaged. Thus, RAS may be the most frequently activated oncoprotein in cancer. We have developed a series of novel, direct, RAS inhibitors with a unique binding site on RAS. The binding of the agents alters the structure of the RAS effector loop and blocks the association with RAS downstream effectors. We used in silico library screening followed by Medicinal Chemistry optimization to develop the compounds. We have validated the agents using Microscale Thermophoresis and NMR to quantify binding to recombinant RAS protein. We have used mutant and wild type RAS driven tumor cell lines in 3D growth and protein-based RAS signaling assays to quantify and characterize the action of the family of inhibitors. We have used xenograft assays to demonstrate compound anti-tumor activity in RAS driven cell lines and pdx systems. We have identified compounds that can bind mutant and wild type K, H, N and M-RAS. We can suppress the association of mutant and wild type RAS protein with its effector RAF-1 in treated cells. The compounds can suppress 3D growth of mutant or wild type RAS driven tumor cell lines and repress tumor development in vivo. The compound family bind to a different site than either clinical agent AMG-510 (K-RAS G12C specific) or MRTX-1133 (K-RAS G12D specific). They exhibit distinct RAS signal inhibition patterns compared to these agents. They can also co-operate with the clinical agents and reduce drug resistance effects. Citation Format: Tariq Arshad, Howard Donninger, Goeff Clarke, Joe Burlison, Rob Monsen, Mike Sabo. In vivo and in vitro experience with novel direct Pan-RAS inhibitors [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 3320.

Oxygen-Bridged Cu Binuclear Sites for Efficient Electrocatalytic CO2 Reduction to Ethanol at Ultralow Overpotential.

Electrocatalytic CO2 reduction (CO2RR) to alcohols offers a promising strategy for converting waste CO2 into valuable fuels/chemicals but usually requires large overpotentials. Herein, we report a catalyst comprising unique oxygen-bridged Cu binuclear sites (CuOCu-N4) with a Cu···Cu distance of 3.0-3.1 Å and concomitant conventional Cu-N4 mononuclear sites on hierarchical nitrogen-doped carbon nanocages (hNCNCs). The catalyst exhibits a state-of-the-art low overpotential of 0.19 V (versus reversible hydrogen electrode) for ethanol and an outstanding ethanol Faradaic efficiency of 56.3% at an ultralow potential of -0.30 V, with high-stable Cu active-site structures during the CO2RR as confirmed by operando X-ray adsorption fine structure characterization. Theoretical simulations reveal that CuOCu-N4 binuclear sites greatly enhance the C-C coupling at low potentials, while Cu-N4 mononuclear sites and the hNCNC support increase the local CO concentration and ethanol production on CuOCu-N4. This study provides a convenient approach to advanced Cu binuclear site catalysts for CO2RR to ethanol with a deep understanding of the mechanism.

A Big Data architecture for early identification and categorization of dark web sites

The dark web has become notorious for its association with illicit activities and there is a growing need for systems to automate the monitoring of this space. This paper proposes an end-to-end scalable architecture for the continuous early identification of new Tor sites and the daily analysis of their content. The solution is built using an Open Source Big Data stack for data serving with Kubernetes, Kafka, Kubeflow, and MinIO, continuously discovering onion addresses in different sources (threat intelligence, code repositories, web-Tor gateways, and Tor repositories), downloading the HTML from Tor and deduplicating the content using MinHash LSH, and categorizing with the BERTopic modeling (SBERT embedding, UMAP dimensionality reduction, HDBSCAN document clustering and c-TF-IDF topic keywords). In 93 days, the system identified 80,049 onion services and characterized 90% of them, addressing the challenge of Tor volatility. A disproportionate amount of repeated content is found, with only 6.1% unique sites. From the HTML files of the dark sites, 31 different low-topics are extracted, manually labeled, and grouped into 11 high-level topics. The five most popular included sexual and violent content, repositories and search engines, carding, cryptocurrencies, and marketplaces. During the experiments, we identified 14 sites with 13,946 clones that shared a suspiciously similar mirroring rate per day, suggesting an extensive common phishing network. Among the related works, this study is the most representative characterization of onion services based on topics to date.

Outstanding Universal Value of World Natural Heritage Sites: Definition Issues

The paper provides an in-depth analysis of the concept of outstanding universal value of world heritage sites, which is the supporting category of the entire system of protection of unique natural sites. The paper considers the normative basis for the definition of this concept in the Convention for the Protection of the World Cultural and Natural Heritage, the Guidelines for its implementation, the practice of the World Heritage Committee and the acts of its advisory bodies. The tripartite division of the composition of outstanding universal value is critically analyzed and the reasons for its appearance are highlighted. Methods of formalizing the analyzed concept are explored, shortcomings in the formulation of wordings of outstanding universal value are highlighted, and various ways to improve this institution are proposed. The comparative legal method of research is widely used: all the problems discussed in the paper are illustrated by the current law enforcement practice of the World Heritage Committee in reviewing the state of conservation of unique natural objects located in foreign countries. The results obtained are compared with the problems of protecting Russian World Natural Heritage sites.

Enhancing seafood traceability: tracking the origin of seabass and seabream from the tuscan coast area by the analysis of the gill bacterial communities

BackgroundThe seafood consumption and trade have increased over the years, and along its expected expansion pose major challenges to the seafood industry and government institutions. In particular, the global trade in fish products and the consequent consumption are linked to reliable authentication, necessary to guarantee lawful trade and healthy consumption. Alterations or errors in this process can lead to commercial fraud and/or health threats. Consequently, the development of new investigative tools became crucial in ensuring unwanted scenarios. Here we used NGS techniques through targeted metagenomics approach on the V3-V4 region of the 16S rRNA genes to characterize the gill bacterial communities in wild-caught seabream (Sparus aurata) and seabass (Dicentrarchus labrax) within different fisheries areas of the “Costa degli Etruschi’’ area in the Tuscan coast. Our challenge involved the possibility of discriminating between the microbiota of both fish species collected from three different fishing sites very close to each other (all within 100 km) in important areas from a commercial and tourist point of view.ResultsOur results showed a significant difference in the assembly of gill bacterial communities in terms of diversity (alpha and beta diversity) of both seabass and seabream in accordance with the three fishing areas. These differences were represented by a unique site -related bacterial signature, more evident in seabream compared to the seabass. Accordingly, the core membership of seabream specimens within the three different sites was minimal compared to the seabass which showed a greater number of sequence variants shared among the different fishing sites. Therefore, the LRT analysis highlighted the possibility of obtaining specific fish bacterial signatures associated with each site; it is noteworthy that specific taxa showed a unique association with the fishing site regardless of the fish species. This study demonstrates the effectiveness of target-metagenomic sequencing of gills in discriminating bacterial signatures of specimens collected from fishing areas located at a limited distance to each other.ConclusionsThis study provides new information relating the structure of the gill microbiota of seabass and seabream in a fishing area with a crucial commercial and tourist interest, namely “Costa degli Etruschi”. This study demonstrated that microbiome-based approaches can represent an important tool for validating the seafood origins with a central applicative perspective in the seafood traceability system.

Investigation of porcine circovirus type 2 and porcine circovirus type 3 infections based on dual TaqMan fluorescent quantitative PCR method and genetic evolutionary analysis of these two viruses.

Porcine circovirus type 2 (PCV2) is the pathogen of Porcine Circovirus Associated Diseases. Porcine circovirus type 3 (PCV3) is a novel porcine circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2 is clearly pathogenic, while the pathogenicity of PCV3 remains controversial, so it is crucial to monitor the prevalence of PCV2 and PCV3 in healthy and diseased pigs to investigate the effects of PCV3 and PCV2 on the health status of pigs. Here, we developed a PCV2 and PCV3 dual TaqMan quantitative PCR (qPCR) method to test samples from healthy and diseased pigs, to clarify the differences in the positive rates and viral copy numbers of PCV2 and PCV3, and to analyze the genetic evolution and molecular characterization of the viral genomes obtained with sequence alignment and phylogenetic analysis, homology and structural analysis of Cap proteins, and selection pressure analysis. We successfully established a dual TaqMan qPCR method for PCV2 and PCV3 with good repeatability, specificity and sensitivity. In total, 1,385 samples from 15 Chinese provinces were tested with the established qPCR. The total positive rates were 37.47% for PCV3 and 57.95% for PCV2, and the coinfection rate for was 25.49%. The positive rates of PCV3 and PCV2 in 372 healthy pigs were 15.05 and 69.89%, respectively, and the coinfection rate was 12.90%. The positive rates of PCV3 and PCV2 in 246 diseased pigs were 55.69 and 83.33%, respectively, and the coinfection rate was 47.97%. Eighteen PCV3 genomes and 64 PCV2 genomes were identified, including nine each of the PCV3a-1 and PCV3b genotypes, eight of PCV2a, 16 of PCV2b, and 40 of PCV2d. The amino acid identity within the PCV3 Cap proteins was 94.00-100.0%, whereas the PCV2 Cap proteins showed an identity of 81.30-100.0%. PCV3 Cap was most variable at amino acid sites 24, 27, 77, 104 and 150, whereas PCV2 Cap had 10-13 unique sites of variation between genotypes. These results clarify the prevalence and variations of PCV2 and PCV3 in healthy and diseased pigs, which will provide a basis for the prevention and control of the two viral infections.

C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription

BackgroundAcute myeloid leukemia (AML) with biallelic (CEBPAbi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs.MethodsHuman AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents.ResultsWe found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo.ConclusionOur results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPAbi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers.

Classification of likely functional class for ligand binding sites identified from fragment screening

Fragment screening is used to identify binding sites and leads in drug discovery, but it is often unclear which binding sites are functionally important. Here, data from 37 experiments, and 1309 protein structures binding to 1601 ligands were analysed. A method to group ligands by binding sites is introduced and sites clustered according to profiles of relative solvent accessibility. This identified 293 unique ligand binding sites, grouped into four clusters (C1-4). C1 includes larger, buried, conserved, and population missense-depleted sites, enriched in known functional sites. C4 comprises smaller, accessible, divergent, missense-enriched sites, depleted in functional sites. A site in C1 is 28 times more likely to be functional than one in C4. Seventeen sites, which to the best of our knowledge are novel, in 13 proteins are identified as likely to be functionally important with examples from human tenascin and 5-aminolevulinate synthase highlighted. A multi-layer perceptron, and K-nearest neighbours model are presented to predict cluster labels for ligand binding sites with an accuracy of 96% and 100%, respectively, so allowing functional classification of sites for proteins not in this set. Our findings will be of interest to those studying protein-ligand interactions and developing new drugs or function modulators.

Computationally inferred cell-type specific epigenome-wide DNA methylation analysis unveils distinct methylation patterns among immune cells for HIV infection in three cohorts.

Epigenome-wide association studies (EWAS) have identified CpG sites associated with HIV infection in blood cells in bulk, which offer limited knowledge of cell-type specific methylation patterns associated with HIV infection. In this study, we aim to identify differentially methylated CpG sites for HIV infection in immune cell types: CD4+ T-cells, CD8+ T-cells, B cells, Natural Killer (NK) cells, and monocytes. Applying a computational deconvolution method, we performed a cell-type based EWAS for HIV infection in three independent cohorts (Ntotal = 1,382). DNA methylation in blood or in peripheral blood mononuclear cells (PBMCs) was profiled by an array-based method and then deconvoluted by Tensor Composition Analysis (TCA). The TCA-computed CpG methylation in each cell type was first benchmarked by bisulfite DNA methylation capture sequencing in a subset of the samples. Cell-type EWAS of HIV infection was performed in each cohort separately and a meta-EWAS was conducted followed by gene set enrichment analysis. The meta-analysis unveiled a total of 2,021 cell-type unique significant CpG sites for five inferred cell types. Among these inferred cell-type unique CpG sites, the concordance rate in the three cohorts ranged from 96% to 100% in each cell type. Cell-type level meta-EWAS unveiled distinct patterns of HIV-associated differential CpG methylation, where 74% of CpG sites were unique to individual cell types (false discovery rate, FDR <0.05). CD4+ T-cells had the largest number of unique HIV-associated CpG sites (N = 1,624) compared to any other cell type. Genes harboring significant CpG sites are involved in immunity and HIV pathogenesis (e.g. CD4+ T-cells: NLRC5, CX3CR1, B cells: IFI44L, NK cells: IL12R, monocytes: IRF7), and in oncogenesis (e.g. CD4+ T-cells: BCL family, PRDM16, monocytes: PRDM16, PDCD1LG2). HIV-associated CpG sites were enriched among genes involved in HIV pathogenesis and oncogenesis that were enriched among interferon-α and -γ, TNF-α, inflammatory response, and apoptotic pathways. Our findings uncovered computationally inferred cell-type specific modifications in the host epigenome for people with HIV that contribute to the growing body of evidence regarding HIV pathogenesis.

Discovering High Entropy Alloy Electrocatalysts in Vast Composition Spaces with Multiobjective Optimization.

High entropy alloys (HEAs) are a highly promising class of materials for electrocatalysis as their unique active site distributions break the scaling relations that limit the activity of conventional transition metal catalysts. Existing Bayesian optimization (BO)-based virtual screening approaches focus on catalytic activity as the sole objective and correspondingly tend to identify promising materials that are unlikely to be entropically stabilized. Here, we overcome this limitation with a multiobjective BO framework for HEAs that simultaneously targets activity, cost-effectiveness, and entropic stabilization. With diversity-guided batch selection further boosting its data efficiency, the framework readily identifies numerous promising candidates for the oxygen reduction reaction that strike the balance between all three objectives in hitherto unchartered HEA design spaces comprising up to 10 elements.

Enabling equitable and ethical research partnerships in crisis situations: Lessons learned from post-disaster heritage protection interventions following Nepal’s 2015 earthquake

The earthquakes which struck Nepal’s capital in 2015 were humanitarian disasters. Not only did they inflict tragic loss of life and livelihoods, they also destroyed parts of the Kathmandu Valley’s unique UNESCO World Heritage site. These monuments were not just ornate structures but living monuments playing central roles in the daily lives of thousands, representing portals where the heavens touch earth and people commune with guiding deities. Their rehabilitation was also of economic importance as they represent a major source of tourist income and employment. Unfortunately, the social and political desire for rapid reconstruction resulted in the swift removal of many traditionally constructed foundations and their replacement with modern materials without assessments of whether they contributed towards the collapse of individual monuments. These actions, combined with the wholesale removal, mixing and dumping of modern and historic debris, contributed to a second, equally destructive, cultural catastrophe – irreversible damage to Kathmandu’s Medieval fabric, in a process which frequently excluded local communities and custodians. This case study draws from our collective reflections and lessons learned from our attempts to enable equitable and ethical research partnerships between UK and Nepali colleagues as well as local communities in the debris of the Kasthamandap, Kathmandu’s eponymous monument. After briefly describing the potential of mobilising archaeologists in post-disaster contexts and outlining the challenges of undertaking research in such a setting, our case study utilises the TRUST Code to assess the character and success of our multidisciplinary collaboration in a time of crises.