Antimicrobial Protein LECT2-b Helps Maintain Gut Microbiota Homeostasis via Selectively Targeting Certain Pathogenic Bacteria.

Antimicrobial peptides/proteins (AMPs) constitute a critical component of gut immunity in animals, protecting the gut from pathogenic bacteria. However, the interactions between AMPs and gut microbiota remain elusive. In this study, we show that leukocyte-derived chemotaxin-2 (LECT2)-b, a recently discovered AMP, helps maintain gut homeostasis in grass carp (Ctenopharyngodon idella), one of the major farmed fish species globally, by directly regulating the gut microbiota. Knockdown of LECT2-b resulted in dysregulation of the gut microbiota. Specifically, LECT2-b deficiency led to the dominance of Proteobacteria, consisting of proinflammatory bacterial species, over Firmicutes, which includes anti-inflammatory bacteria. In addition, the opportunistic pathogenic bacteria genus Aeromonas became the dominant genus replacing the probiotic bacteria Lactobacillus and Bacillus. Further analysis revealed that this effect was due to the direct and selective inhibition of certain pathogenic bacterial species by LECT2-b. Moreover, LECT2-b knockdown promoted biofilm formation by gut microbiota, resulting in tissue damage and inflammation. Importantly, LECT2-b treatment alleviated the negative effects induced by LECT2-b knockdown. These findings highlight the crucial role of LECT2-b in maintaining the gut microbiota homeostasis and mucosal health. Overall, our study provides important data for understanding the roles of AMPs in the regulation of gut homeostasis in animals.

An Atlas of Grass Carp IgM+ B Cells in Homeostasis and Bacterial Infection Helps to Reveal the Unique Heterogeneity of B Cells in Early Vertebrates.

Teleost B cells are primitive lymphocytes with both innate and adaptive immune functions. However, the heterogeneity and differentiation trajectory of teleost B cells remain largely unknown. In this study, the landscape of grass carp IgM+ (gcIgM+) B cells was revealed by single-cell RNA sequencing. The results showed that gcIgM+ B cells mainly comprise six populations: (im)mature B cells, innate B cells, proliferating B cells, plasma cells, CD22+ cells, and CD34+ cells, among which innate B cells and proliferating B cells were uncommon B cell subsets with, to our knowledge, new characteristics. Remarkably, three functional IgMs were discovered in grass carp, and a significant percentage of gcIgM+ B cells, especially plasma cells, expressed multiple Igµ genes (Igµ1, Igµ2, and/or Igµ3). More importantly, through single-cell sorting combined with Sanger sequencing, we found that distinct VHDJH recombination patterns of Igµ genes were present in single IgM+ B cells, indicating that individual teleost B cells might produce multiple Abs by coexpressing rearranged IgM subclass genes. Moreover, the percentage of IgM1highIgM2highIgM3high plasma cells increased significantly after bacterial infection, suggesting that individual plasma cells might tend to produce multiple IgMs to resist the infection in teleost fish. In summary, to our knowledge, this study not only helps to uncover the unique heterogeneity of B cells in early vertebrates but also provided significant new evidence supporting the recently proposed "one cell-multiple Abs" paradigm, challenging the classical rule of "one cell-one Ab."

Identification and functional analyses of CD4-1+ cells in grass carp (Ctenopharyngodon idella).

In mammals, CD4 is found to be expressed on T cells and innate immune cells, however, teleost cells bearing CD4 have not been well identified and characterized. In this study, we identified two different CD4-1+ cell subsets in grass carp (Ctenopharyngodon idella): CD4-1+ lymphocytes (Lym) and CD4-1+ myeloid cells (Mye), both of which had the highest proportions in the head kidney. The mRNA expression analysis showed that CD4-1, CD4-2, TCRß, CD3γ/δ, and LCK1 are highly expressed in CD4-1+ Lym and also expressed in CD4-1+ Mye. Furthermore, we found that CD4-1+ Lym have a Lym morphology and highly express T-cell cytokines, suggesting that they are CD4+ T cells equivalent to mammalian Th cells. On the other hand, CD4-1+ Mye were found to have a morphology of macrophage and highly express macrophage marker gene MCSFR, indicating that they are macrophages. In addition, functional analysis revealed that CD4-1+ Mye possess phagocytic ability and great antigen-processing ability. Taken together, our study sheds further light on the composition and function of CD4+ cells in teleost fish.

LECT2 Is a Novel Antibacterial Protein in Vertebrates.

In vertebrates, leukocyte-derived chemotaxin-2 (LECT2) is an important immunoregulator with conserved chemotactic and phagocytosis-stimulating activities to leukocytes during bacterial infection. However, whether LECT2 possesses direct antibacterial activity remains unknown. In this article, we show that, unlike tetrapods with a single LECT2 gene, two LECT2 genes exist in teleost fish, named LECT2-a and LECT2-b Using grass carp as a research model, we found that the expression pattern of grass carp LECT2-a (gcLECT2-a) is more similar to that of LECT2 in tetrapods, while gcLECT2-b has evolved to be highly expressed in mucosal immune organs, including the intestine and skin. Interestingly, we found that gcLECT2-b, with conserved chemotactic and phagocytosis-stimulating activities, can also kill Gram-negative and Gram-positive bacteria directly in a membrane-dependent and a non-membrane-dependent manner, respectively. Moreover, gcLECT2-b could prevent the adherence of bacteria to epithelial cells through agglutination by targeting peptidoglycan and lipoteichoic acid. Further study revealed that gcLECT2-b can protect grass carp from Aeromonas hydrophila infection in vivo, because it significantly reduces intestinal necrosis and tissue bacterial load. More importantly, we found that LECT2 from representative tetrapods, except human, also possesses direct antibacterial activities, indicating that the direct antibacterial property of LECT2 is generally conserved in vertebrates. Taken together, to our knowledge, our study discovered a novel function of LECT2 in the antibacterial immunity of vertebrates, especially teleost fish, greatly enhancing our knowledge of this important molecule.

Insights into the Antibacterial Properties of Complement Peptides C3a, C4a, and C5a across Vertebrates.

Complement peptides C3a, C4a, and C5a are important components of innate immunity in vertebrates. Although they diverged from a common ancestor, only C3a and C4a can act as antibacterial peptides in Homo sapiens, suggesting that C5a has evolved into a purely chemotactic molecule; however, the antibacterial properties of C3a, C4a, and C5a across vertebrates still require elucidation. In this article, we show that, unlike those in H. sapiens, Mus musculus C3a, C4a, and C5a all possess antibacterial activities, implying that the antibacterial properties of C3a, C4a, and C5a have evolved divergently in vertebrates. The extremely different net charge, a key factor determining the antibacterial activities of cationic antimicrobial peptides, of vertebrate C3a, C4a, and C5a supports this speculation. Moreover, the antibacterial activity of overlapping peptides covering vertebrate C3a, C4a, and C5a further strongly supports the speculation, because their activity is positively correlated with the net charge of source molecules. Notably, the structures of C3a, C4a, and C5a are conserved in vertebrates, and the inactive overlapping peptides can become antibacterial peptides if mutated to possess enough net positive charges, indicating that net charge is the only factor determining the antibacterial properties of vertebrate C3a, C4a, and C5a. More importantly, many vertebrate C3a-, C4a-, and C5a-derived peptides possess high antibacterial activities yet exhibit no hemolytic activities, suggesting the application potential in anti-infective therapy. Taken together, our findings reveal that vertebrate C3a, C4a, and C5a are all sources of antibacterial peptides that will facilitate the design of excellent peptide antibiotics.

Insights into the Antibacterial Properties of Complement Peptides C3a, C4a, and C5a across Vertebrates.

Complement peptides C3a, C4a, and C5a are important components of innate immunity in vertebrates. Although they diverged from a common ancestor, only C3a and C4a can act as antibacterial peptides in Homo sapiens, suggesting that C5a has evolved into a purely chemotactic molecule; however, the antibacterial properties of C3a, C4a, and C5a across vertebrates still require elucidation. In this article, we show that, unlike those in H. sapiens, Mus musculus C3a, C4a, and C5a all possess antibacterial activities, implying that the antibacterial properties of C3a, C4a, and C5a have evolved divergently in vertebrates. The extremely different net charge, a key factor determining the antibacterial activities of cationic antimicrobial peptides, of vertebrate C3a, C4a, and C5a supports this speculation. Moreover, the antibacterial activity of overlapping peptides covering vertebrate C3a, C4a, and C5a further strongly supports the speculation, because their activity is positively correlated with the net charge of source molecules. Notably, the structures of C3a, C4a, and C5a are conserved in vertebrates, and the inactive overlapping peptides can become antibacterial peptides if mutated to possess enough net positive charges, indicating that net charge is the only factor determining the antibacterial properties of vertebrate C3a, C4a, and C5a. More importantly, many vertebrate C3a-, C4a-, and C5a-derived peptides possess high antibacterial activities yet exhibit no hemolytic activities, suggesting the application potential in anti-infective therapy. Taken together, our findings reveal that vertebrate C3a, C4a, and C5a are all sources of antibacterial peptides that will facilitate the design of excellent peptide antibiotics.

Post-sepsis psychiatric disorder: Pathophysiology, prevention, and treatment.

Post-sepsis psychiatric disorder, encompassing anxiety, depression, post-traumatic stress disorder and delirium, is a highly prevalent complication secondary to sepsis, resulting in a marked increase in long-term mortality among affected patients. Regrettably, psychiatric impairment associated with sepsis is frequently disregarded by clinicians. This review aims to summarize recent advancements in the understanding of the pathophysiology, prevention, and treatment of post-sepsis mental disorder, including coronavirus disease 2019-related psychiatric impairment. The pathophysiology of post-sepsis psychiatric disorder is complex and is known to involve blood-brain barrier disruption, overactivation of the hypothalamic-pituitary-adrenal axis, neuroinflammation, oxidative stress, neurotransmitter dysfunction, programmed cell death, and impaired neuroplasticity. No unified diagnostic criteria for this disorder are currently available; however, screening scales are often applied in its assessment. Modifiable risk factors for psychiatric impairment post-sepsis include the number of experienced traumatic memories, the length of ICU stay, level of albumin, the use of vasopressors or inotropes, daily activity function after sepsis, and the cumulative dose of dobutamine. To contribute to the prevention of post-sepsis psychiatric disorder, it may be beneficial to implement targeted interventions for these modifiable risk factors. Specific therapies for this condition remain scarce. Nevertheless, non-pharmacological approaches, such as comprehensive nursing care, may provide a promising avenue for treating psychiatric disorder following sepsis. In addition, although several therapeutic drugs have shown preliminary efficacy in animal models, further confirmation of their potential is required through follow-up clinical studies.

Preparation of the monoclonal antibody against Nile tilapia Igλ and study on the Igλ+ B cell subset in Nile tilapia.

Immunoglobulins (Igs) are important effector molecules that mediate humoral immunity. A typical Ig consists of two heavy and two light chains. In teleosts, three Ig heavy chain isotypes (Igµ, Igδ and Igτ) and three Ig light chain isotypes (Igκ, Igλ and Igσ) have been identified. Compared to the heavy chains, teleost Ig light chains have been poorly studied due to the lack of antibodies. In this study, a mouse anti-Nile tilapia Igλ monoclonal antibody (mAb) was prepared, which could specifically recognize Igλ in serum and Igλ+ B cells in tissues. Further, the composition of IgM+ and Igλ+ B cell subsets was analyzed using this antibody and a mouse anti-tilapia IgM heavy chain mAb. The ratio of IgM+Igλ+ B cells to total IgM+ B cells in head kidney and peripheral blood was about 30%, while that in spleen was about 50%; the ratio of IgM-Igλ+ B cells to total Igλ+ B cells in head kidney and peripheral blood was about 45%, while that in spleen was about 25%. The IgM-Igλ+ B cells was speculated to be IgT+ B cells. Finally, we detected an increase in the level of specific antibodies against the surface antigen-Sip of Streptococcus agalactiae in serum after S. agalactiae infection, indicating that mouse anti-tilapia Igλ mAb can be used to detect the antibody level after immunization of Nile tilapia, which lays a foundation for the evaluation of immunization effect of tilapia vaccine.

A novel N-heterocycles substituted oseltamivir derivatives as potent inhibitors of influenza virus neuraminidase: discovery, synthesis and biological evaluation.

Our previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop.

Combination of canagliflozin and puerarin alleviates the lipotoxicity to diabetic kidney in mice.

Diabetic kidney disease is one of the most serious complications of diabetes. Although diabetic kidney disease can be effectively controlled through strict blood glucose management and corresponding symptomatic treatment, these therapies cannot reduce its incidence in diabetic patients. The sodium-glucose cotransporter 2 (SGLT2) inhibitors and the traditional Chinese herb "Gegen" have been widely used in diabetes-related therapy. However, it remains unclear whether the combined use of these two kinds of medicines contributes to an increased curative effect on diabetic kidney disease. In this study, we examined this issue by evaluating the efficacy of the combination of puerarin, an active ingredient of Gegen, and canagliflozin, an SGLT2 inhibitor for a 12-week intervention using a mouse model of diabetes. The results indicated that the combination of puerarin and canagliflozin was superior to canagliflozin alone in improving the metabolic and renal function parameters of diabetic mice. Our findings suggested that the renoprotective effect of combined puerarin and canagliflozin in diabetic mice was achieved by reducing renal lipid accumulation. This study provides a new strategy for the clinical prevention and treatment of diabetic kidney disease. The puerarin and SGLT2 inhibitor combination therapy at the initial stage of diabetes may effectively delay the occurrence of diabetic kidney injury, and significantly alleviate the burden of renal lipotoxicity.

Chromosome-level genome assembly of grass carp (Ctenopharyngodon idella) provides insights into its genome evolution.

BACKGROUND: The grass carp has great economic value and occupies an important evolutionary position. Genomic information regarding this species could help better understand its rapid growth rate as well as its unique body plan and environmental adaptation. RESULTS: We assembled the chromosome-level grass carp genome using the PacBio sequencing and chromosome structure capture technique. The final genome assembly has a total length of 893.2 Mb with a contig N50 of 19.3 Mb and a scaffold N50 of 35.7 Mb. About 99.85% of the assembled contigs were anchored into 24 chromosomes. Based on the prediction, this genome contained 30,342 protein-coding genes and 43.26% repetitive sequences. Furthermore, we determined that the large genome size can be attributed to the DNA-mediated transposable elements which accounted for 58.9% of the repetitive sequences in grass carp. We identified that the grass carp has only 24 pairs of chromosomes due to the fusion of two ancestral chromosomes. Enrichment analyses of significantly expanded and positively selected genes reflected evolutionary adaptation of grass carp to the feeding habits. We also detected the loss of conserved non-coding regulatory elements associated with the development of the immune system, nervous system, and digestive system, which may be critical for grass carp herbivorous traits. CONCLUSIONS: The high-quality reference genome reported here provides a valuable resource for the genetic improvement and molecular-guided breeding of the grass carp.

Discovery of novel 1,2,4-triazole phenylalanine derivatives targeting an unexplored region within the interprotomer pocket of the HIV capsid protein.

Human immunodeficiency virus (HIV) capsid (CA) protein is a promising target for developing novel anti-HIV drugs. Starting from highly anticipated CA inhibitors PF-74, we used scaffold hopping strategy to design a series of novel 1,2,4-triazole phenylalanine derivatives by targeting an unexplored region composed of residues 106-109 in HIV-1 CA hexamer. Compound d19 displayed excellent antiretroviral potency against HIV-1 and HIV-2 strains with EC50 values of 0.59 and 2.69 µM, respectively. Additionally, we show via surface plasmon resonance (SPR) spectrometry that d19 preferentially interacts with the hexameric form of CA, with a significantly improved hexamer/monomer specificity ratio (ratio = 59) than PF-74 (ratio = 21). Moreover, we show via SPR that d19 competes with CPSF-6 for binding to CA hexamers with IC50 value of 33.4 nM. Like PF-74, d19 inhibits the replication of HIV-1 NL4.3 pseudo typed virus in both early and late stages. In addition, molecular docking and molecular dynamics simulations provide binding mode information of d19 to HIV-1 CA and rationale for improved affinity and potency over PF-74. Overall, the lead compound d19 displays a distinct chemotype form PF-74, improved CA affinity, and anti-HIV potency.

Indolylarylsulfones bearing phenylboronic acid and phenylboronate ester functionalities as potent HIV­1 non-nucleoside reverse transcriptase inhibitors.

To explore the chemical space around the entrance channel of the HIV-1 reverse transcriptase (RT) binding pocket, we innovatively designed and synthesized a series of novel indolylarylsulfones (IASs) bearing phenylboronic acid and phenylboronate ester functionalities at the indole-2-carboxamide as new HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) through structure-based drug design. All the newly synthesized compounds exhibited excellent to moderate potency against wild-type (WT) HIV-1 with EC50 values ranging from 6.7 to 42.6 nM. Among all, (3-ethylphenyl)boronic acid substituted indole-2-carboxamide and (4-ethylphenyl) boronate ester substituted indole-2-carboxamide were found to be the most potent inhibitors (EC50 = 8.5 nM, SI = 3310; EC50 = 6.7 nM, SI = 3549, respectively). Notably, (3-ethylphenyl)boronic acid substituted indole-2-carboxamide maintained excellent activities against the single HIV-1 mutants L100I (EC50 = 7.3 nM), K103N (EC50 = 9.2 nM), as well as the double mutant V106A/F227L (EC50 = 21.1 nM). Preliminary SARs and molecular modelling studies are also discussed in detail.

Design, synthesis, and biological evaluation of novel sulfamoylbenzamide derivatives as HBV capsid assembly modulators.

Capsid assembly modulators (CAMs) represent a novel class of antiviral agents targeting hepatitis B virus (HBV) capsid to disrupt the assembly process. NVR 3-778 is the first CAM to demonstrate antiviral activity in patients infected with HBV. However, the relatively low aqueous solubility and moderate activity in the human body halted further development of NVR 3-778. To improve the anti-HBV activity and the drug-like properties of NVR 3-778, we designed and synthesized a series of NVR 3-778 derivatives. Notably, phenylboronic acid-bearing compound 7b (EC50 = 0.83 ± 0.33 µM, CC50 = 19.4 ± 5.0 µM) displayed comparable anti-HBV activity to NVR 3-778 (EC50 = 0.73 ± 0.20 µM, CC50 = 23.4 ± 7.0 µM). Besides, 7b showed improved water solubility (328.8 µg/mL, pH 7) compared to NVR 3-778 (35.8 µg/mL, pH 7). Size exclusion chromatography (SEC) and quantification of encapsidated viral RNA were used to demonstrate that 7b behaves as a class II CAM similar to NVR 3-778. Moreover, molecular dynamics (MD) simulations were conducted to rationalize the structure-activity relationships (SARs) of these novel derivatives and to understand their key interactions with the binding pocket, which provide useful indications for guiding the further rational design of more effective anti-HBV drugs.

Bearing Fault Diagnosis Based on Randomized Fisher Discriminant Analysis.

In this paper, a novel randomized Fisher discriminant analysis (RFDA) based bearing fault diagnosis method is proposed. First, several representative time-domain features are extracted from the raw vibration signals. Second, linear Fisher discriminant analysis (FDA) is extended to nonlinear FDA named RFDA by introducing the random feature map to deal with the non-linearity issue. Specifically, the extracted time-domain features data are mapped onto a high-dimensional space using the random feature map function rather than kernel functions. Third, the time-domain features are fed into the built RFDA model to extract the discriminant features for diagnosis. Moreover, a Bayesian inference is employed to identify the class of the collected vibration signals to diagnose the bearing status. The proposed method uses random Fourier features to approximate the kernel matrix in the kernel Fisher discriminant analysis. Through employing randomized Fisher discriminant analysis, the nonlinearity issue is dealt with, and the computational burden is remarkably reduced compared to the kernel Fisher discriminant analysis (KFDA). To illustrate the superior performance of the proposed RFDA-based bearing fault diagnosis method, comparative experiments are conducted on two widely used datasets, the Case Western Reserve University (CWRU) bearing dataset and the Paderborn University (PU) bearing dataset. For the CWRU dataset, the computation time of RFDA is much shorter than KFDA, while the accuracy rate reaches the same level of KFDA. For the PU dataset, the accuracy rate of RFDA is slightly higher than KFDA, and the computation time is only 44.14% of KFDA.

Newly Emerging Strategies in Antiviral Drug Discovery: Dedicated to Prof. Dr. Erik De Clercq on Occasion of His 80th Anniversary.

Viral infections pose a persistent threat to human health. The relentless epidemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health problem, with millions of infections and fatalities so far. Traditional approaches such as random screening and optimization of lead compounds by organic synthesis have become extremely resource- and time-consuming. Various modern innovative methods or integrated paradigms are now being applied to drug discovery for significant resistance in order to simplify the drug process. This review provides an overview of newly emerging antiviral strategies, including proteolysis targeting chimera (PROTAC), ribonuclease targeting chimera (RIBOTAC), targeted covalent inhibitors, topology-matching design and antiviral drug delivery system. This article is dedicated to Prof. Dr. Erik De Clercq, an internationally renowned expert in the antiviral drug research field, on the occasion of his 80th anniversary.

Discovery and Mechanistic Investigation of Piperazinone Phenylalanine Derivatives with Terminal Indole or Benzene Ring as Novel HIV-1 Capsid Modulators.

HIV-1 capsid (CA) performs multiple roles in the viral life cycle and is a promising target for antiviral development. In this work, we describe the design, synthesis, assessment of antiviral activity, and mechanistic investigation of 20 piperazinone phenylalanine derivatives with a terminal indole or benzene ring. Among them, F2-7f exhibited moderate anti-HIV-1 activity with an EC50 value of 5.89 µM, which was slightly weaker than the lead compound PF74 (EC50 = 0.75 µM). Interestingly, several compounds showed a preference for HIV-2 inhibitory activity, represented by 7f with an HIV-2 EC50 value of 4.52 µM and nearly 5-fold increased potency over anti-HIV-1 (EC50 = 21.81 µM), equivalent to PF74 (EC50 = 4.16 µM). Furthermore, F2-7f preferred to bind to the CA hexamer rather than to the monomer, similar to PF74, according to surface plasmon resonance results. Molecular dynamics simulation indicated that F2-7f and PF74 bound at the same site. Additionally, we computationally analyzed the ADMET properties for 7f and F2-7f. Based on this analysis, 7f and F2-7f were predicted to have improved drug-like properties and metabolic stability over PF74, and no toxicities were predicted based on the chemotype of 7f and F2-7f. Finally, the experimental metabolic stability results of F2-7f in human liver microsomes and human plasma moderately correlated with our computational prediction. Our findings show that F2-7f is a promising small molecule targeting the HIV-1 CA protein with considerable development potential.

Design, Synthesis, and Mechanistic Study of 2-Pyridone-Bearing Phenylalanine Derivatives as Novel HIV Capsid Modulators.

The AIDS pandemic is still of importance. HIV-1 and HIV-2 are the causative agents of this pandemic, and in the absence of a viable vaccine, drugs are continually required to provide quality of life for infected patients. The HIV capsid (CA) protein performs critical functions in the life cycle of HIV-1 and HIV-2, is broadly conserved across major strains and subtypes, and is underexploited. Therefore, it has become a therapeutic target of interest. Here, we report a novel series of 2-pyridone-bearing phenylalanine derivatives as HIV capsid modulators. Compound FTC-2 is the most potent anti-HIV-1 compound in the new series of compounds, with acceptable cytotoxicity in MT-4 cells (selectivity index HIV-1 > 49.57; HIV-2 > 17.08). However, compound TD-1a has the lowest EC50 in the anti-HIV-2 assays (EC50 = 4.86 ± 1.71 µM; CC50= 86.54 ± 29.24 µM). A water solubility test found that TD-1a showed a moderately increased water solubility compared with PF74, while the water solubility of FTC-2 was improved hundreds of times. Furthermore, we use molecular simulation studies to provide insight into the molecular contacts between the new compounds and HIV CA. We also computationally predict drug-like properties and metabolic stability for FTC-2 and TD-1a. Based on this analysis, TD-1a is predicted to have improved drug-like properties and metabolic stability over PF74. This study increases the repertoire of CA modulators and has important implications for developing anti-HIV agents with novel mechanisms, especially those that inhibit the often overlooked HIV-2.

Design, Synthesis, and Evaluation of a Set of Carboxylic Acid and Phosphate Prodrugs Derived from HBV Capsid Protein Allosteric Modulator NVR 3-778.

Hepatitis B virus (HBV) capsid protein (Cp) is necessary for viral replication and the maintenance of viral persistence, having become an attractive target of anti-HBV drugs. To improve the water solubility of HBV capsid protein allosteric modulator (CpAM) NVR 3-778, a series of novel carboxylic acid and phosphate prodrugs were designed and synthesized using a prodrug strategy. In vitro HBV replication assay showed that these prodrugs maintained favorable antiviral potency (EC50 = 0.28−0.42 µM), which was comparable to that of NVR 3-778 (EC50 = 0.38 µM). More importantly, the cytotoxicity of prodrug N8 (CC50 > 256 µM) was significantly reduced compared to NVR 3-778 (CC50 = 13.65 ± 0.21 µM). In addition, the water solubility of prodrug N6 was hundreds of times better than that of NVR 3-778 in three phosphate buffers with various pH levels (2.0, 7.0, 7.4). In addition, N6 demonstrated excellent plasma and blood stability in vitro and good pharmacokinetic properties in rats. Finally, the hemisuccinate prodrug N6 significantly improved the candidate drug NVR 3-778's water solubility and increased metabolic stability while maintaining its antiviral efficacy.

Insights into activated sludge/Chlorella consortia under dark condition compared with light condition.

Bacteria-algae consortia in the light bring the benefit of O2 production and CO2 reduction for wastewater treatment, while the bottleneck for application is how it behaves in the dark. In this study, inoculum ratio and sludge retention time (SRT) affected nutrient removal rather than chemical oxygen demand (COD) removal. Dark conditions (with a sludge/Chlorella inoculum ratio of 1:2 at a SRT of 15 d) achieved comparable performance to those of light conditions, due to bacteria contribution and mechanical aeration. Compared with light conditions, the ratio of Chla/Chlb decreased and Caro/(Chla + Chlb) increased to response oxidative stress. In the dark, algae were associated with Nitrosomonas and Dechloromonas. Flavobacterium disassociated with Chlorella in the dark but associated with Chlorella in the light. Moreover, nitritation genes (amo and Hao) and denitrifying gene (narH) were up-regulated, while P metabolism genes (PPX and PPK) were down-regulated. It is proposed to enrich Nitrosomonas in the night and denitrify polyphosphate accumulating organisms (DPAO) in the daytime to establish short-cut nitrification and denitrifying phosphorus removal in practical applications.