Machine learning-guided design of potent darunavir analogs targeting HIV-1 proteases: A computational approach for antiretroviral drug discovery.

In the pursuit of novel antiretroviral therapies for human immunodeficiency virus type-1 (HIV-1) proteases (PRs), recent improvements in drug discovery have embraced machine learning (ML) techniques to guide the design process. This study employs ensemble learning models to identify crucial substructures as significant features for drug development. Using molecular docking techniques, a collection of 160 darunavir (DRV) analogs was designed based on these key substructures and subsequently screened using molecular docking techniques. Chemical structures with high fitness scores were selected, combined, and one-dimensional (1D) screening based on beyond Lipinski's rule of five (bRo5) and ADME (absorption, distribution, metabolism, and excretion) prediction implemented in the Combined Analog generator Tool (CAT) program. A total of 473 screened analogs were subjected to docking analysis through convolutional neural networks scoring function against both the wild-type (WT) and 12 major mutated PRs. DRV analogs with negative changes in binding free energy ( ΔΔ G bind ) compared to DRV could be categorized into four attractive groups based on their interactions with the majority of vital PRs. The analysis of interaction profiles revealed that potent designed analogs, targeting both WT and mutant PRs, exhibited interactions with common key amino acid residues. This observation further confirms that the ML model-guided approach effectively identified the substructures that play a crucial role in potent analogs. It is expected to function as a powerful computational tool, offering valuable guidance in the identification of chemical substructures for synthesis and subsequent experimental testing.

Nucleic Acid-Templated Synthesis of Cationic Styryl Dyes in Vitro and in Living Cells.

A novel method for synthesizing cationic styryl dyes through a nucleic acid-templated reaction has been developed. This approach overcomes issues associated with traditional synthesis methods, such as harsh conditions, low throughput, and wasteful chemicals. The presence of a nucleic acid template accelerated the styryl dye formation from quaternized heteroaromatic and cationic aldehyde substrates. These styryl dyes show remarkable optical properties change when bound to nucleic acids, hence the success of the synthesis could be readily monitored in situ by UV-Vis and fluorescence spectroscopy and the optical properties data were also observable at the same time. This method provides the desired products from a broad range of coupling partners. By employing different substrates and templates, it is possible to identify new dyes that can bind to a specific type of nucleic acid such as a G-quadruplex. The templated dye synthesis is also successfully demonstrated in live HeLa cells. This approach is a powerful tool for the rapid synthesis and screening of dyes specific for diverse types of nucleic acids or cellular organelles, facilitating new biological discoveries.

Effect of NH4Cl supplementation on growth, photosynthesis, triacylglycerol content in Chlamydomonas reinhardtii under mixotrophic cultivation.

AIM: Ammonium chloride (NH4Cl) is one of the nitrogen sources for microalgal cultivation. An excessive amounts of NH4Cl are toxic for microalgae. However, combining mixotrophic conditions and excessive quantities of NH4Cl positively affects microalgal biomass and lipid production. In this study, we investigated the impact of NH4Cl on the growth, biomass, and triglyceride (TAG) content of the green microalga Chlamydomonas reinhardtii especially under mixotrophic condition. METHODS AND RESULTS: Under photoautotrophic conditions (without organic carbon supplementation), adding 25 mM NH4Cl had no significant effect on microalgal growth or TAG content. However, under mixotrophic condition (with acetate supplementation), NH4Cl interfered with microalgal growth while inducing TAG content. To explore these effects further, we conducted a two-step cultivation process and found that NH4Cl reduced microalgal growth, but induced total lipid and TAG content, especially after 4-day cultivation. The photosynthesis performances showed that NH4Cl completely inhibited oxygen evolution on Day 4. However, NH4Cl slightly reduced the Fv/Fm ratio indicating that the NH4Cl supplementation directly affects microalgal photosynthesis. To investigate the TAG induction effect by NH4Cl, we compared the protein expression profiles of microalgae grown mixotrophically with and without 25 mM NH4Cl using a proteomics approach. This analysis identified 1,782 proteins, with putative acetate uptake transporter GFY5 and Acyl-coenzyme A oxidase being overexpressed in the NH4Cl-treated group. CONCLUSION: These findings suggested that NH4Cl supplementation may stimulate acetate utilization and fatty acid synthesis pathways in microalgae cells. Our study indicated that NH4Cl supplementation can induce microalgal biomass and lipid production, particularly when combined with mixotrophic conditions.

A quest for novel antimicrobial targets: Inhibition of Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) by synthetic analogs of aminoacyl-adenosine in vitro and live bacteria.

The Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) has been proposed as a novel antibacterial drug target due to its indispensability in prominent human pathogens. While several inhibitors with in vitro activity have been identified, none have been demonstrated to have potent activity against live bacteria. In this work, seven non-hydrolyzable transition state mimics of GatCAB were synthesized and tested as the transamidase inhibitors against GatCAB from the human pathogen Helicobacter pylori. Notably, the methyl sulfone analog of glutamyl-adenosine significantly reduced GatCAB's transamination rate. Additionally, four lipid-conjugates of these mimics displayed antibacterial activity against Bacillus subtilis, likely due to enhanced cell permeability. Inhibitory activity against GatCAB in live bacteria was confirmed using a sensitive gain-of-function dual luciferase reporter in Mycobacterium bovis-BCG. Only the lipid-conjugated methyl sulfone analog exhibited a significant increase in mistranslation rate, highlighting its cell permeability and inhibitory potential. This study provides insights for developing urgently needed novel antibacterial agents amidst emerging antimicrobial drug resistance.

Reduction of surface treatment time by combination of citric acid and ascorbic acid while restoring shear bond strength of metal brackets bonded to bleached enamel: a pilot study.

BACKGROUND: To investigate the effect of a 50% ascorbic acid with 50% citric acid solution on the immediate shear bond strength (SBS) of metallic brackets after tooth bleaching. The enamel etching pattern and the required quantity of these combined acids as antioxidants following 35% hydrogen peroxide (HP) bleaching were also determined. METHODS: The stability of the solution at room temperature was assessed at various time intervals. Fifty teeth were randomly divided into five groups: non-bleached (G1), bleached then acid etched (G2), bleached followed by a 10-minute treatment with 10% sodium ascorbate and acid etched (G3), 5-minute treatment with 50% ascorbic acid (G4), and 5-minute treatment with a combination of 50% ascorbic acid and 50% citric acid (G5). Groups G2, G3, G4 and G5 were bleached by 35% HP gel for a total of 32 min. Acid etching in groups G1, G2, and G3 was performed using 37% phosphoric acid (Ormco®, Orange, CA, USA) for 15 s. In all groups, metal brackets were immediately bonded using Transbond™ XT primer and Transbond™ PLUS adhesive, with light curing for 40 s. The SBS was tested with a universal testing machine, and statistical analysis was conducted using one-way ANOVA followed by Tukey's HSD test. The level of significance was set at p < 0.05 for all statistical tests. RESULTS: Stability tests demonstrated that the combined acids remained effective for up to 21 days. Group G5 significantly increased the SBS of bleached teeth to the level of G1 (p < 0.05), while G3 did not achieve the same increase in SBS (p > 0.05). SEM analysis revealed enamel etching patterns similar to those of both control groups (G1 and G2). Kinetic studies at 6 min indicated that the antioxidation in G5 reacted 0.2 mmole lower than in G3 and G4. CONCLUSION: 5-minute application of the combined acids enhanced the SBS of bleached teeth comparable to unbleached teeth. The combined acids remain stable over two weeks, presenting a time-efficient, single-step solution for antioxidant application and enamel etching in orthodontic bracket bonding.

Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase.

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.

Possibility to Apply Strontium Aluminate to Produce Light-Emitting Plants: Efficiency and Safety.

Light-emitting plants (LEPs) provides light in areas without electricity. The phosphorescent compound was used as a lighting material for LEP development. However, using the phosphorescent compound for LEPs development required optimization and phytotoxicity evaluation. Strontium aluminate (SrAl2 O4 ) is a phosphorescent compound that can glow for a long time and is easily recharged by visible light. In this study, using SrAl2 O4 to develop LEPs was evaluated. Additionally, plant stress under SrAl2 O4 was investigated. Metabolomic analysis can explain the possible mechanism of plants' stress under SrAl2 O4 . After, injecting 3 mL of 5 % (w/v) SrAl2 O4 products 1, 2, and 3 into the stem of Ipomoea aquatica, the result showed that SrAl2 O4 products 2 and 3 caused oxidative stress. The metabolomic analysis also indicated that I. aquatica responded to SrAl2 O4 product 1 by increasing pipecolic acid and salicylic acid, while I. aquatica injected with SrAl2 O4 products 2 and 3 showed a decrease in salicylic acid around 0.005 and 0.061-fold, respectively, compared to control plants. and an excess accumulation of MDA around 10.00-12.00 µmol g-1 FW. A 15 % concentration of SrAl2 O4 can be used for LEPs development, enabling photoemission 18-fold for 50 min. SrAl2 O4 product 1 has the potential to be a material for LEPs.

Exploring the Apoptotic-Induced Biochemical Mechanism of Traditional Thai Herb (Kerra™) Extract in HCT116 Cells Using a Label-Free Proteomics Approach.

Background and Objectives: Natural products have proven to be a valuable source for the discovery of new candidate drugs for cancer treatment. This study aims to investigate the potential therapeutic effects of "Kerra™", a natural extract derived from a mixture of nine medicinal plants mentioned in the ancient Thai scripture named the Takxila Scripture, on HCT116 cells. Materials and Methods: In this study, the effect of the Kerra™ extract on cancer cells was assessed through cell viability assays. Apoptotic activity was evaluated by examining the apoptosis characteristic features. A proteomics analysis was conducted to identify proteins and pathways associated with the extract's mechanism of action. The expression levels of apoptotic protein markers were measured to validate the extract's efficacy. Results: The Kerra™ extract demonstrated a dose-dependent inhibitory effect on the cells, with higher concentrations leading to decreased cell viability. Treatment with the extract for 72 h induced characteristic features of early and late apoptosis, as well as cell death. An LC-MS/MS analysis identified a total of 3406 proteins. The pathway analysis revealed that the Kerra™ extract stimulated apoptosis and cell death in colorectal cancer cell lines and suppressed cell proliferation in adenocarcinoma cell lines through the EIF2 signaling pathway. Upstream regulatory proteins, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and MYC proto-oncogene, bHLH transcription factor (MYC), were identified. The expressions of caspase-8 and caspase-9 were significantly elevated by the Kerra™ extract compared to the chemotherapy drug Doxorubicin (Dox). Conclusions: These findings provide strong evidence for the ability of the Kerra™ extract to induce apoptosis in HCT116 colon cancer cells. The extract's efficacy was demonstrated by its dose-dependent inhibitory effect, induction of apoptotic activity, and modulation of key proteins involved in cell death and proliferation pathways. This study highlights the potential of Kerra™ as a promising therapeutic agent in cancer treatment.

Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection.

O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

Optogenetic control of kinetochore function.

Kinetochores act as hubs for multiple activities during cell division, including microtubule interactions and spindle checkpoint signaling. Each kinetochore can act autonomously, and activities change rapidly as proteins are recruited to, or removed from, kinetochores. Understanding this dynamic system requires tools that can manipulate kinetochores on biologically relevant temporal and spatial scales. Optogenetic approaches have the potential to provide temporal and spatial control with molecular specificity. Here we report new chemical inducers of protein dimerization that allow us to both recruit proteins to and release them from kinetochores using light. We use these dimerizers to manipulate checkpoint signaling and molecular motor activity. Our findings demonstrate specialized properties of the CENP-E (kinesin-7) motor for directional chromosome transport to the spindle equator and for maintenance of metaphase alignment. This work establishes a foundation for optogenetic control of kinetochore function, which is broadly applicable to experimental probing of other dynamic cellular processes.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer.

Many dynamic biological processes are regulated by protein-protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on subcellular levels by controlling protein-protein interactions.

Protein kinase R-like endoplasmatic reticulum kinase is a mediator of stretch in ventilator-induced lung injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe form of lung injury characterized by damage to the epithelial barrier with subsequent pulmonary edema and hypoxic respiratory failure. ARDS is a significant medical problem in intensive care units with associated high care costs. There are many potential causes of ARDS; however, alveolar injury associated with mechanical ventilation, termed ventilator-induced lung injury (VILI), remains a well-recognized contributor. It is thus critical to understand the mechanism of VILI. Based on our published preliminary data, we hypothesized that the endoplasmic reticulum (ER) stress response molecule Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) plays a role in transmitting mechanosensory signals the alveolar epithelium. METHODS: ER stress signal responses to mechanical stretch were studied in ex-vivo ventilated pig lungs. To explore the effect of PERK inhibition on VILI, we ventilated live rats and compared lung injury parameters to non-ventilated controls. The effect of stretch-induced epithelial ER Ca2+ signaling on PERK was studied in stretched alveolar epithelial monolayers. To confirm the activation of PERK in human disease, ER stress signaling was compared between ARDS and non-ARDS lungs. RESULTS: Our studies revealed increased PERK-specific ER stress signaling in response to overstretch. PERK inhibition resulted in dose-dependent improvement of alveolar inflammation and permeability. Our data indicate that stretch-induced epithelial ER Ca2+ release is an activator of PERK. Experiments with human lung tissue confirmed PERK activation by ARDS. CONCLUSION: Our study provides evidences that PERK is a mediator stretch signals in the alveolar epithelium.

Protective Effects of an Octapeptide Identified from Riceberry™ (Oryza sativa) Protein Hydrolysate on Oxidative and Endoplasmic Reticulum (ER) Stress in L929 Cells.

Reactive oxygen species (ROS) play a critical role in oxidative stress and cellular damage, underscoring the importance of identifying potent antioxidants. This research focuses on the antioxidant capabilities of Riceberry™-derived peptides and their protective effects against oxidative and endoplasmic reticulum (ER) stress in L929 cells. By simulating human digestion, Riceberry™ protein hydrolysate was generated, from which antioxidant peptides were isolated using OFFGEL electrophoresis and LC-MS/MS. Notably, an octapeptide (VPAGVAHW) from the hydrolysate demonstrated significant antioxidant activity, particularly against oxidative stress induced by iodoacetic acid (IAA) or hydrogen peroxide (H2O2) and ER stress caused by tunicamycin (TM) in L929 cells. This peptide's effectiveness was evident in its dose-dependent ability to enhance cell viability and mitigate stress effects, although its efficiency varied with the stress inducer. Our study suggests that Riceberry™-derived peptides could serve as a promising natural antioxidant with potential benefits for health promotion and applications in the food industry, offering an environmentally friendly alternative to synthetic antioxidants.

Vernonia amygdalina Leaf Extract Induces Apoptosis in HeLa Cells: A Metabolomics and Proteomics Study.

Medicinal plants produce various bioactive molecules with potential anti-cancer properties with favorable safety profiles. We aimed to investigate the comprehensive composition of Vernonia amygdalina leaf extract and its cytotoxic effects via apoptosis in HeLa cells. The metabolomics approach using LC-MS/MS was conducted to gather the metabolite profile of the extract. Proteomics was performed to understand the comprehensive mechanistic pathways of action. The apoptosis was visualized by cellular staining and the apoptotic proteins were evaluated. V. amygdalina leaf extract exhibited dose-dependent cytotoxic effects on both HeLa and Vero cells after 24 h of exposure in the MTT assay with the IC50 values of 0.767 ± 0.0334 and 4.043 ± 0.469 µg mL-1, respectively, which demonstrated a higher concentration required for Vero cell cytotoxicity. The metabolomic profile of 112 known metabolites specified that the majority of them were alkaloids, phenolic compounds, and steroids. Among these metabolites, deacetylvindoline and licochalcone B were suggested to implicate cytotoxicity. The cytotoxic pathways involved the response to stress and cell death which was similar to doxorubicin. The upstream regulatory proteins, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and X-box binding protein 1 (XBP1), were significantly altered, supporting the regulation of apoptosis and cell death. The levels of apoptotic proteins, c-Jun N-terminal kinases (JNK), p53, and caspase-9 were significantly increased. The novel insights gained from the metabolomic profiling and proteomic pathway analysis of V. amygdalina leaf extract have identified crucial components related to apoptosis induction, highlighting its potential to develop future chemotherapy.

A Novel Bacitracin-like Peptide from Mangrove-Isolated Bacillus paralicheniformis NNS4-3 against MRSA and Its Genomic Insights.

The global rise of antimicrobial resistance (AMR) presents a critical challenge necessitating the discovery of novel antimicrobial agents. Mangrove microbes are valuable sources of new antimicrobial compounds. This study reports the discovery of a potent antimicrobial peptide (AMP) from Bacillus paralicheniformis NNS4-3, isolated from mangrove sediment, exhibiting significant activity against methicillin-resistant Staphylococcus aureus (MRSA). The AMP demonstrated a minimum inhibitory concentration ranging from 1 to 16 µg/mL in the tested bacteria and exhibited bactericidal effects at higher concentrations. Structural analysis revealed a bacitracin-like configuration and the peptide acted by disrupting bacterial membranes in a time- and concentration-dependent manner. The AMP maintained stability under heat, proteolytic enzymes, surfactants, and varying pH treatments. The ten biosynthetic gene clusters (BGCs) of secondary metabolites were found in the genome. Detailed sequence comparison of the predicted bacitracin BGC indicated distinct DNA sequences compared to previously reported strains. Although the antibiotic resistance genes were found, this strain was susceptible to antibiotics. Our findings demonstrated the potential of Bacillus paralicheniformis NNS4-3 and its AMP as a promising agent in combating AMR. The genetic information could be pivotal for future applications in the healthcare industry, emphasizing the need for continued exploration of marine microbial diversity in drug discovery.

FMO-guided design of darunavir analogs as HIV-1 protease inhibitors.

The prevalence of HIV-1 infection continues to pose a significant global public health issue, highlighting the need for antiretroviral drugs that target viral proteins to reduce viral replication. One such target is HIV-1 protease (PR), responsible for cleaving viral polyproteins, leading to the maturation of viral proteins. While darunavir (DRV) is a potent HIV-1 PR inhibitor, drug resistance can arise due to mutations in HIV-1 PR. To address this issue, we developed a novel approach using the fragment molecular orbital (FMO) method and structure-based drug design to create DRV analogs. Using combinatorial programming, we generated novel analogs freely accessible via an on-the-cloud mode implemented in Google Colab, Combined Analog generator Tool (CAT). The designed analogs underwent cascade screening through molecular docking with HIV-1 PR wild-type and major mutations at the active site. Molecular dynamics (MD) simulations confirmed the assess ligand binding and susceptibility of screened designed analogs. Our findings indicate that the three designed analogs guided by FMO, 19-0-14-3, 19-8-10-0, and 19-8-14-3, are superior to DRV and have the potential to serve as efficient PR inhibitors. These findings demonstrate the effectiveness of our approach and its potential to be used in further studies for developing new antiretroviral drugs.

A new benzoquinone and a new stilbenoid from Paphiopedilum exul (Ridl.) Rolfe.

One new alkyl benzoquinone, paphionone (1), one new trans-stilbenoid, (E)-6,5'-dihydroxy-2,3'-dimethoxystilbene (2), and eight known stilbenoids and flavonoids (3-10) were isolated from the leaves and roots of Paphiopedilum exul (Orchidaceae). Their chemical structures were determined based on IR, ECD, MS and NMR analyses. Cytotoxicity of all isolated compounds towards human hepatocellular carcinoma (HepG2) cell line was examined in vitro by MTT assay. The para-hydroxybenzyl substituted stilbene 10 was potently cytotoxic to the cancer cells, with an IC50 value of 4.80 ± 1.10 µM (selectivity index = 20.83). All compounds were non-toxic to normal human embryo fibroblast (OUMS-36) cell line.

A phage nucleus-associated protein from the jumbophage Churi inhibits bacterial growth through protein translation interference.

Antibacterial proteins inhibiting Pseudomonas aeruginosa have been identified in various phages and explored as antibiotic alternatives. Here, we isolated a phiKZ-like phage, Churi, which encodes 364 open reading frames. We examined 15 early-expressed phage proteins for their ability to inhibit bacterial growth, and found that gp335, closely related to phiKZ-gp14, exhibits antibacterial activity. Similar to phiKZ-gp14, recently shown to form a complex with the P. aeruginosa ribosome, we predict experimentally that gp335 interacts with ribosomal proteins, suggesting its involvement in protein translation. GFP-tagged gp335 clusters around the phage nucleus as early as 15 minutes post-infection and remains associated with it throughout the infection, suggesting its role in protein expression in the cell cytoplasm. CRISPR-Cas13-mediated deletion of gp355 reveals that the mutant phage has a prolonged latent period. Altogether, we demonstrate that gp335 is an antibacterial protein of nucleus-forming phages that associates with the ribosomes at the phage nucleus.