Inhibition Of Protein Synthesis Research Articles

Chemical Modification of Pactamycin Leads to New Compounds with Retained Antimicrobial Activity and Reduced Toxicity

Pactamycin (PCT), an antibiotic produced by Streptomyces pactum, is a five-membered ring aminocyclitol that is active against a variety of Gram-positive and Gram-negative microorganisms, as well as several animal tumor lines in culture and in vivo. Pactamycin targets the small ribosomal subunit and inhibits protein synthesis in bacteria, archaea, and eukaryotes, but due to its toxicity is used only as a tool for biochemical research. Prompted by the successful and well-established procedure for the derivatization of antibiotics, we modified pactamycin by tethering basic amino acids to the free primary amino group of the aminocyclitol ring. Specifically, lysine, ornithine, and histidine were conjugated via an amide bond, and the antimicrobial activity of the derivatives was evaluated both in vivo and in vitro. According to our results, their antimicrobial activity was maintained at almost equal levels, while their toxicity was reduced compared to the parent molecule. These findings suggest that the new pactamycin derivatives can be considered as promising pharmacophores for the development of new antimicrobials that are able to combat the dangerously increasing resistance of pathogens to antibiotics.

Differentiation activates mitochondrial OPA1 processing in myoblast cell lines

Mitochondrial optic atrophy-1 (OPA1) plays key roles in adapting mitochondrial structure to bioenergetic function. When transmembrane potential across the inner membrane (Δψm) is intact, long (L-OPA1) isoforms shape the inner membrane through membrane fusion and the formation of cristal junctions. When Δψm is lost, however, OPA1 is cleaved to short, inactive S-OPA1 isoforms by the OMA1 metalloprotease, disrupting mitochondrial structure and priming cellular stress responses such as apoptosis. Previously, we demonstrated that L-OPA1 of H9c2 cardiomyoblasts is insensitive to loss of Δψm via challenge with the protonophore carbonyl cyanide chlorophenyl hydrazone (CCCP), but that CCCP-induced OPA1 processing is activated upon differentiation in media with low serum supplemented with all-trans retinoic acid (ATRA). Here, we show that this developmental induction of OPA1 processing in H9c2 cells is independent of ATRA; moreover, pretreatment of undifferentiated H9c2s with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, recapitulates the Δψm-sensitive OPA1 processing observed in differentiated H9c2s. L6.C11 and C2C12 myoblast lines display the same developmental and CAP-sensitive induction of OPA1 processing, demonstrating a general mechanism of OPA1 regulation in mammalian myoblast cell settings. Restoration of CCCP-induced OPA1 processing correlates with increased apoptotic sensitivity. Moreover, OPA1 knockdown indicates that intact OPA1 is necessary for effective myoblast differentiation. Taken together, our results indicate that a novel developmental mechanism acts to regulate OMA1-mediated OPA1 processing in myoblast cell lines, in which differentiation engages mitochondrial stress sensing.

Defects in the central metabolism prevent thymineless death in Escherichia coli, while still allowing significant protein synthesis.

Starvation of E. coli thyA auxotrophs for the required thymine or thymidine leads to cessation of DNA synthesis and, unexpectedly, to thymineless death (TLD). Previously, TLD-alleviating defects were identified by the candidate gene approach, for their contribution to replication initiation, fork repair, or SOS induction. However, no TLD-blocking mutations were ever found, suggesting a multifactorial nature of TLD. Since (until recently) no unbiased isolation of TLD suppressors was reported, we used enrichment after insertional mutagenesis to systematically isolate TLD suppressors. Our approach was validated by isolation of known TLD-alleviating mutants in recombinational repair. At the same time, and unexpectedly for the current TLD models, most of the isolated suppressors affected general metabolism, while the strongest suppressors impacted the central metabolism. Several temperature-sensitive (Ts)-mutants in important/essential functions, like nadA, ribB or coaA, almost completely suppressed TLD at 42°C. Since blocking protein synthesis completely by chloramphenicol prevents TLD, while reducing protein synthesis to 10% alleviates TLD only slightly, we measured the level of protein synthesis in these mutants at 42°C and found it to be 20-70% of the WT, not enough reduction to explain TLD prevention. We conclude that the isolated central metabolism mutants prevent TLD by affecting specific TLD-promoting functions.

The fungal natural product fusidic acid demonstrates potent activity against Mycoplasma genitalium.

Antimicrobial resistance is extremely common in Mycoplasma genitalium, a frequent cause of urethritis in men and cervicitis, vaginitis, and pelvic inflammatory disease in women. Treatment of M. genitalium infections is difficult due to intrinsic and acquired resistance to many antibiotic classes. We undertook a program to identify novel antimicrobials with activity against M. genitalium from fungal natural products. Extracts of Ramularia coccinea contained a molecule with potent activity that was subsequently identified as fusidic acid, a fusidane-type antibiotic that has been in clinical use for decades outside the United States. We found that minimum inhibitory concentrations of fusidic acid ranged from 0.31 to 4 µg/mL among 17 M. genitalium strains including laboratory-passaged and low-passage clinical isolates. Time-kill data indicate that bactericidal killing occurs when M. genitalium is exposed to ≥10 µg/mL for 48 h, comparing favorably to serum concentrations obtained from typical loading dose regimens. Resistance to fusidic acid was associated with mutations in fusA consistent with the known mechanism of action in which fusidic acid inhibits protein synthesis by binding to elongation factor G. Interestingly, no mutants resistant to >10 µg/mL fusidic acid were obtained and a resistant strain containing a F435Y mutation in FusA was impaired for growth in vitro. These data suggest that fusidic acid may be a promising option for the treatment of M. genitalium infections.

Antiviral effect of the viroporin inhibitors against Taiwan isolates of infectious bronchitis virus (IBV)

Coronaviruses (CoVs) are significant animal and human pathogens, characterized by being enveloped RNA viruses with positive-sense single-stranded RNA. The Coronaviridae family encompasses four genera, among which gammacoronaviruses pose a major threat to the poultry industry, which infectious bronchitis virus (IBV) being the most prominent of these threats. Particularly, IBV adversely affects broiler growth and egg production, causing substantial losses. The IBV strains currently circulating in Taiwan include the IBV Taiwan-I (TW-I) serotype, IBV Taiwan-II (TW-II) serotype, and vaccine strains. Therefore, ongoing efforts have focused on developing novel vaccines and discovering antiviral agents. The envelope (E) proteins of CoVs accumulate in the endoplasmic reticulum-Golgi intermediate compartment prior to virus budding. These E proteins assemble into viroporins, exhibiting ion channel activity that leads to cell membrane disruption, making them attractive targets for antiviral therapy.In this study, we investigated the E proteins of IBV H-120, as well as IBV serotypes TW-I and TW-II. E protein expression resulted in inhibited bacteria growth, increased permeability of bacteria to β-galactosidase substrates, and blocked protein synthesis of bacteria by hygromycin B (HygB). Furthermore, in the presence of E proteins, HygB also impeded protein translation in DF-1 cells and damaged their membrane integrity. Collectively, these findings confirm the viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. Next, the viroporin inhibitors, 5-(N,N-hexamethylene) amiloride (HMA) and 4,4’-diisothiocyano stilbene-2,2’-disulphonic acid (DIDS) were used to inhibit the viroporin activities of the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. In chicken embryos and chickens infected with IBV serotypes TW-I and IBV TW-II, no survivors were observed at 6 and 11 days post-infection (dpi), respectively. However, treatments with both DIDS and HMA increased the survival rates in infected chicken embryos and chickens and mitigated histopathological lesions in the trachea and kidney. Additionally, a 3D pentameric structure of the IBV E protein was constructed via homology modeling. As expected, both inhibitors were found to bind to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction. Taken together, our findings provide comprehensive evidence supporting the use of viroporin inhibitors as promising antiviral agents against IBV Taiwan isolates.

Restriction of arginine induces antibiotic tolerance in Staphylococcus aureus

Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

Fear generalization modulated by shock intensity and protein synthesis inhibitor.

Maladaptive fear responses, including sensitized threat reactions and overgeneralization, contribute to anxiety disorders such as generalized anxiety disorder and post-traumatic stress disorder. Although stress intensity influences the generation and extent of these maladaptive fears, the underlying mechanisms remain unclear. The present study examined whether varying footshock stress intensity and inhibition of protein synthesis have differential effect on fear sensitization and generalization in mice. Mice were subjected to a classic fear conditioning protocol involving five different levels of footshock intensities. Prior to fear acquisition, the protein synthesis inhibitor cycloheximide (CHX) was administered intraperitoneally. Fear sensitization to white noise and fear generalization to tones with frequencies differing from the conditioned tone were assessed at either 2 or 4 days after fear acquisition. The results showed that, although varying shock intensities (except the lowest) led to a similar pattern of increased freezing during auditory cues in fear acquisition, the extent of both fear sensitization and generalization increased with the intensity of the footshock in the following days. As shock intensities increased, there was a proportional rise in sensitized fear to white noise and generalized freezing to tones with frequencies progressively closer to the conditioned stimulus. Mildest shocks did not induce discriminative conditioned fear memory, whereas the most intense shocks led to pronounced fear generalization. Administration of CHX before fear acquisition did not affect sensitized fear but reduced generalization of freezing to tones dissimilar from the conditioned stimulus in the group exposed to the most intense shock. Our results suggest that maladaptive fear responses elicited by varying stress intensities exhibit distinct characteristics. The effect of CHX to prevent overgeneralization without affecting discriminative fear memory points to potential therapeutic approaches for fear-related disorders, suggesting the possibility of mitigating overgeneralization while preserving necessary fear discrimination.

Suppressing Mycobacterium tuberculosis virulence and drug resistance by targeting Eis protein through computational drug discovery.

Tuberculosis (TB) remains a critical health threat, particularly with the emergence of multidrug-resistant strains. This demands attention from scientific communities and healthcare professionals worldwide to develop effective treatments. The enhanced intracellular survival (Eis) protein is an acetyltransferase enzyme of Mycobacterium tuberculosis that functions by adding acetyl groups to aminoglycoside antibiotics, which interferes with their ability to bind to the bacterial ribosome, thereby preventing them from inhibiting protein synthesis and killing the bacterium. Therefore, targeting this protein accelerates the chance of restoring the aminoglycoside drug activity, thereby reducing the emergence of drug-resistant TB. For this, we have screened 406,747 natural compounds from the Coconut database against Eis protein. Based on MM/GBSA rescoring binding energy, the top 5 most prominent natural compounds, viz. CNP0187003 (-96.14kcal/mol), CNP0176690 (-93.79kcal/mol), CNP0136537 (-92.31kcal/mol), CNP0398701 (-91.96kcal/mol), and CNP0043390 (-91.60kcal/mol) were selected. These compounds exhibited the presence of a substantial number of hydrogen bonds and other significant interactions confirming their strong binding affinity with the Eis protein during the docking process. Subsequently, the MD simulation of these compounds exhibited that the Eis-CNP0043390 complex was the most stable, followed by Eis-CNP0187003 and Eis-CNP0176690 complex, further verified by binding free energy calculation, principal component analysis (PCA), and Free energy landscape analysis. These compounds demonstrated the most favourable results in all parameters utilised for this investigation and may have the potential to inhibit the Eis protein. There these findings will leverage computational techniques to identify and develop a natural compound inhibitor as an alternative for drug-resistant TB.

Antibacterial and mechanism of action studies of boxazomycin A.

Boxazomycins A-C are potent broad-spectrum antibiotics isolated from Actinomycetes strain G495-1 in 1987. We now report that boxazomycin A inhibits bacterial growth by selectively inhibiting protein synthesis, its effect is bacteriostatic, and it is equally active against drug resistant bacterial strains. No cross-resistance to protein synthesis inhibitors was observed suggesting that its inhibition is distinct from clinical protein synthesis inhibitors. We also report in vivo efficacy in a Staphylococcus aureus murine infection model supported by corresponding pharmacokinetic studies.

Amidino-rocaglates (ADRs), a class of synthetic rocaglates, are potent inhibitors of SARS-CoV-2 replication through inhibition of viral protein synthesis

Coronaviruses are highly transmissible respiratory viruses that cause symptoms ranging from mild congestion to severe respiratory distress. The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need for new antivirals with broad-acting mechanisms to combat increasing emergence of new variants. Currently, there are only a few antivirals approved for treatment of SARS-CoV-2. Previously, the rocaglate natural product silvestrol and synthetic rocaglates such as CR-1-31b were shown to have antiviral effects by inhibiting eukaryotic translation initiation factor 4A1 (eIF4A) function and virus protein synthesis. In this study, we evaluated amidino-rocaglates (ADRs), a class of synthetic rocaglates with the most potent eIF4A-inhibitory activity to-date, for inhibition of SARS-CoV-2 infection. This class of compounds showed low nanomolar potency against multiple SARS-CoV-2 variants and in multiple cell types, including human lung-derived cells, with strong inhibition of virus over host protein synthesis and low cytotoxicity. The most potent ADRs were also shown to be active against two highly pathogenic and distantly related coronaviruses, SARS-CoV and MERS-CoV. Mechanistically, cells with mutations of eIF4A1, which are known to reduce rocaglate interaction displayed reduced ADR-associated loss of cellular function, consistent with targeting of protein synthesis. Overall, ADRs and derivatives may offer new potential treatments for SARS-CoV-2 with the goal of developing a broad-acting anti-coronavirus agent.

Targeting the redox vulnerability of acute myeloid leukaemia cells with a combination of auranofin and vitamin C.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by complex molecular and cytogenetic abnormalities. Pro-oxidant cellular redox status is a common hallmark of AML cells, providing a rationale for redox-based anticancer strategy. We previously discovered that auranofin (AUF), initially used for the treatment of rheumatoid arthritis and repositioned for its anticancer activity, can synergize with a pharmacological concentration of vitamin C (VC) against breast cancer cell line models. In this study, we observed that this drug combination synergistically and efficiently killed cells of leukaemic cell lines established from different myeloid subtypes. In addition to an induced elevation of reactive oxygen species and ATP depletion, a rapid dephosphorylation of 4E-BP1 and p70S6K, together with a strong inhibition of protein synthesis were early events in response to AUF/VC treatment, suggesting their implication in AUF/VC-induced cytotoxicity. Importantly, a study on 22 primary AML specimens from various AML subtypes showed that AUF/VC combinations at pharmacologically achievable concentrations were effective to eradicate primary leukaemic CD34+ cells from the majority of these samples, while being less toxic to normal cord blood CD34+ cells. Our findings indicate that targeting the redox vulnerability of AML with AUF/VC combinations could present a potential anti-AML therapeutic approach.

Exploring synergistic and antagonistic interactions in phage-antibiotic combinations against ESKAPE pathogens.

In the era of antimicrobial resistance, phage-antibiotic combinations offer a promising therapeutic option, yet research on their synergy and antagonism is limited. This study aims to assess these interactions, focusing on protein synthesis inhibitors and cell envelope-active agents against multidrug-resistant bacterial strains. We evaluated synergistic and antagonistic interactions in multidrug-resistant Staphylococcus aureus, Enterococcus faecium, and Pseudomonas aeruginosa strains. Phages were combined with protein synthesis inhibitors [linezolid (LZD), minocycline (MIN), gentamicin (GEN), and azithromycin (AZM)] or cell envelope-active agents [daptomycin (DAP), ceftaroline (CPT), and cefepime (FEP)]. Modified checkerboard minimum inhibitory concentration assays and 24-h time-kill analyses were conducted, alongside one-step growth curves to analyze phage growth kinetics. Statistical comparisons used one-way analysis of variance (ANOVA) and the Tukey test (P < 0.05). In the checkerboard and 24-h time-kill analyses (TKA) of S. aureus and E. faecium, phage-LZD and phage-MIN combinations were antagonistic (FIC > 4) while phage-DAP and phage-CPT were synergistic (FIC 0.5) (ANOVA range of mean differences 0.52-2.59 log10 CFU/mL; P < 0.001). For P. aeruginosa, phage-AZM was antagonistic (FIC > 4), phage-GEN was additive (FIC = 1), and phage-FEP was synergistic (ANOVA range of mean differences 1.04-1.95 log10 CFU/mL; P < 0.001). Phage growth kinetics were altered in the presence of LZD and MIN against S. aureus and in the presence of LZD against a single E. faecium strain (HOU503). Our findings indicate that select protein synthesis inhibitors may induce phage-antibiotic antagonism. However, this antagonism may not solely stem from changes in phage growth kinetics, warranting further investigation into the complex interplay among strains, phage attributes, and antibiotic mechanisms affecting bacterial inhibition.IMPORTANCEIn the face of escalating antimicrobial resistance, combining phages with antibiotics offers a promising avenue for treating infections unresponsive to traditional antibiotics. However, while studies have explored synergistic interactions, less attention has been given to potential antagonism and its impact on phage growth kinetics. This research evaluates the interplay between phages and antibiotics, revealing both synergistic and antagonistic patterns across various bacterial strains and shedding light on the complex dynamics that influence treatment efficacy. Understanding these interactions is crucial for optimizing combination therapies and advancing phage therapy as a viable solution for combating antimicrobial resistance.

African swine fever virus pCP312R interacts with host RPS27A to shut off host protein translation and promotes viral replication

African swine fever virus (ASFV) severely threatens the global economy and food security. ASFV encodes >150 genes, but the functions of most of them have yet to be characterized in detail. Here we explored the function of the ASFV CP312R gene and found that CP312R plays an essential role in ASFV replication. Knockout of the CP312R gene terminated viral replication and CP312R knockdown substantially suppressed ASFV infection in vitro. Furthermore, we resolved the crystal structure of pCP312R to 2.3 Å resolution and found that pCP312R has the potential to bind nucleic acids. LC-MS analysis and co-immunoprecipitation assay revealed that pCP312R interacts with RPS27A, a component of the 40S ribosomal subunit. Confocal microscopy showed the interaction between pCP312R and RPS27A leaded to a modification in the subcellular localization of this host protein, which suppresses host protein translation. Renilla-Glo luciferase assay and Ribopuromycylation analysis evidenced that knockout of RPS27A completely aborted the shutoff activity of pCP312R, and trans-complementation of RPS27A recovered pCP312R shutoff activity in RPS27A-knockout cells. Our findings shed light on the function of ASFV CP312R gene in virus infection, which triggers inhibition of host protein synthesis.

M2 macrophage exosome-derived Apoc1 promotes ferroptosis resistance in osteosarcoma by inhibiting ACSF2 deubiquitination.

Osteosarcoma (OS) is the most common primary malignant tumor of bone. The aim of this study was to investigate the regulatory mechanisms of M2 macrophage exosomes (M2-Exos) in ferroptosis in OS. A mouse model was established to investigate the in vivo role of M2-Exos. We investigated their effects on ferroptosis in OS using erastin, a ferroptosis activator, and deferoxamine mesylate, an iron chelator. In vitro, we investigated whether the Apoc1/Acyl-CoA Synthetase Family Member 2 (ACSF2) axis mediates these effects, using shApoc1 and shACSF2. The mechanisms whereby Apoc1 regulates ACSF2 were examined using cyclohexanone, a protein synthesis inhibitor, and MG132, a proteasomal inhibitor. M2-Exos reversed the inhibitory effects of erastin on OS cells, thus enhancing their viability, migration, invasion, proliferation, and reducing ferroptosis. Apoc1 was highly expressed in M2-Exos, and interfering with this expression reversed the effects of M2-Exos on OS cells. ACSF2 mediated the effects of M2-Exos-derived Apoc1. Apoc1 interacted with ACSF2, which, in turn, interacted with USP40. Apoc1 overexpression increased ACSF2 ubiquitination, promoting its degradation, whereas USP40 overexpression inhibited ACSF2 ubiquitination and promoted its expression. Apoc1 overexpression inhibited ACSF2 binding to USP40. M2-Exos-derived Apoc1 promoted ferroptosis resistance by inhibiting USP40 binding to ACSF2 and promoting ACSF2 ubiquitination and degradation, thus enhancing OS development.

All-trans retinoic acid induces lipophagy by reducing Rubicon in Hepa1c1c7 cells

All-trans retinoic acid (atRA), a metabolite of vitamin A, reduces hepatic lipid accumulation in liver steatosis model animals. Lipophagy, a new lipolysis pathway, degrades a lipid droplet (LD) via autophagy in adipose tissue and the liver. We recently found that atRA induces lipophagy in adipocytes. However, it remains unclear whether atRA induces lipophagy in hepatocytes. In this study, we investigated the effects of atRA on lipophagy in Hepa1c1c7 cells and the liver of mice fed a high-fat diet (HFD). First, we confirmed that atRA induced autophagy in Hepa1c1c7 cells by Western blotting and the GFP-LC3-mCherry probe. Next, we evaluated the lipolysis in fatty Hepa1c1c7 cells treated with the knockdown of Atg5, an essential gene in autophagy induction. Atg5-knockdown partly suppressed the atRA-induced lipolysis in fatty Hepa1c1c7 cells. We also found that atRA reduced the protein, but not mRNA, expression of Rubicon, a negative regulator of autophagy, in Hepa1c1c7 cells and the liver of HFD-fed mice. Rubicon-knockdown partly inhibited the atRA-induced lipolysis in fatty Hepa1c1c7 cells. In addition, atRA reduced hepatic Rubicon expression in young mice, but the effect of atRA on it diminished in aged mice. Finally, we investigated the mechanism underlying reduced Rubicon protein expression by atRA in hepatocytes. A protein synthesis inhibitor, but not proteasome or lysosomal inhibitors, significantly blocked the reduction of Rubicon protein expression by atRA in Hepa1c1c7 cells. These results suggest that atRA may promote lipophagy in fatty hepatocytes by reducing hepatic Rubicon expression via inhibiting protein synthesis.

Induction of long-term hyperexcitability by memory-related cAMP signaling in isolated nociceptor cell bodies.

Persistent hyperactivity of nociceptors is known to contribute significantly to long-lasting sensitization and ongoing pain in many clinical conditions. It is often assumed that nociceptor hyperactivity is mainly driven by continuing stimulation from inflammatory mediators. We have tested an additional possibility: that persistent increases in excitability promoting hyperactivity can be induced by a prototypical cellular signaling pathway long known to induce late-phase long-term potentiation (LTP) of synapses in brain regions involved in memory formation. This cAMP-PKA-CREB-gene transcription-protein synthesis pathway was tested using whole-cell current clamp methods on small dissociated sensory neurons (primarily nociceptors) from dorsal root ganglia (DRGs) excised from previously uninjured ("naïve") rats. Six-hour treatment with the specific Gαs-coupled 5-HT4 receptor agonist, prucalopride, or with the adenylyl cyclase activator, forskolin, induced long-term hyperexcitability (LTH) in DRG neurons that manifested 12-24 hours later as action potential (AP) discharge (ongoing activity, OA) during artificial depolarization to -45 mV, a membrane potential that is normally subthreshold for AP generation. Prucalopride treatment also induced significant long-lasting depolarization of resting membrane potential (from -69 to -66 mV), enhanced depolarizing spontaneous fluctuations (DSFs) of membrane potential, and indications of reduced AP threshold and rheobase. LTH was prevented by co-treatment of prucalopride with inhibitors of PKA, CREB, gene transcription, and protein synthesis. As in the induction of synaptic memory, many other cellular signals are likely to be involved. However, the discovery that this prototypical memory induction pathway can induce nociceptor LTH, along with reports that cAMP signaling and CREB activity in DRGs can induce hyperalgesic priming, suggest that early, temporary, cAMP-induced transcriptional and translational mechanisms can induce nociceptor LTH that might last for long periods. An interesting possibility is that these mechanisms can also be reactivated by re-exposure to inflammatory mediators such as serotonin during subsequent challenges to bodily integrity, "reconsolidating" the cellular memory and thereby extending the duration of persistent nociceptor hyperexcitability.

Acquisition of Resistance to PEGylated Branched Polyethylenimine Increases Pseudomonas Aeruginosa Susceptibility to Aminoglycosides.

PEGylated branched polyethylenimine (PEG-BPEI) has antibacterial and antibiofilm properties. Exposure to PEG-BPEI through serial passage leads to resistant P. aeruginosa strains. The minimum inhibitory concentration (MIC) of 600 Da BPEI and PEGylated 600 Da BPEI (PEG-BPEI) in the wild-type PAO1 strain is 16 μg/ml while, after 15 serial passages, the MIC increased to 1024 μg/mL. An additional 15 rounds of serial passage in the absence of BPEI or PEG-BPEI did not change the 1024 μg/mL MIC. Gentamicin, Neomycin, and Tobramycin, cationic antibiotics that inhibit protein synthesis, have a 16-32 fold reduction of MIC values in PEG350-BPEI resistant strains, suggesting increased permeation. The influx of these antibiotics occurs using a self-mediated uptake mechanism, suggesting changes to the outer membrane Data show that resistance causes changes in genes related to outer membrane lipopolysaccharide (LPS) assembly. Mutations were noted in the gene coding for the polymerase Wzy that participates in the assembly of the O-antigen region. Other mutations were noted with wbpE and wbpI of the Wbp pathway responsible for the enzymatic synthesis of ManNAc(3NAc)A in the LPS of P. aeruginosa. These changes suggest that an altered gene product could lead to PEG-BPEI resistance. Nevertheless, the increased susceptibility to aminoglycosides could prevent the emergence of PEG-BPEI resistant bacterial populations.

Targeting a microbiota Wolbachian aminoacyl-tRNA synthetase to block its pathogenic host.

The interplay between humans and their microbiome is crucial for various physiological processes, including nutrient absorption, immune defense, and maintaining homeostasis. Microbiome alterations can directly contribute to diseases or heighten their likelihood. This relationship extends beyond humans; microbiota play vital roles in other organisms, including eukaryotic pathogens causing severe diseases. Notably, Wolbachia, a bacterial microbiota, is essential for parasitic worms responsible for lymphatic filariasis and onchocerciasis, devastating human illnesses. Given the lack of rapid cures for these infections and the limitations of current treatments, new drugs are imperative. Here, we disrupt Wolbachia's symbiosis with pathogens using boron-based compounds targeting an unprecedented Wolbachia enzyme, leucyl-tRNA synthetase (LeuRS), effectively inhibiting its growth. Through a compound demonstrating anti-Wolbachia efficacy in infected cells, we use biophysical experiments and x-ray crystallography to elucidate the mechanism behind Wolbachia LeuRS inhibition. We reveal that these compounds form adenosine-based adducts inhibiting protein synthesis. Overall, our study underscores the potential of disrupting key microbiota to control infections.

Spatiotemporally Controllable Covalent Bonding of RNA for Multi-Protein Interference.

Many diseases are associated with genetic mutation and expression of mutated proteins, such as cancers. Therapeutic approaches that selectively target the synthesis process of multiple proteins show greater potential compared to single-protein approaches in oncological diseases. However, conventional agents to regulate the synthesis of multiple protein still suffer from poor spatiotemporal selectivity and stability. Here, a new method using a dye-peptide conjugate, PRFK, for multi-protein interference with spatiotemporal selectivity and reliable stability, is reported. By using the peptide sequence that targets tumor cells, PRFK can be efficiently taken up, followed by specific binding to the KDELR (KDEL receptor) proteinlocated in the endoplasmic reticulum (ER). The dye generates 1O2 under light irradiation, enabling photodynamic therapy. This processconverts the furan group into a cytidine-reactive intermediate, whichcovalentlybinds to mRNA, thereby blocking protein synthesis. Upon treating 4T1 cells, the proteomics data show alterations in apoptosis, ferroptosis, proliferation, migration, invasion, and immune infiltration, suggesting that multi-protein interference leads to the disruption of cellular physiological activities, ultimately achieving tumor treatment. This study presents a multi-protein interference probe with the potential for protein interference within various subcellular organelles in the future.

Notopterygium Incisum Extract Promotes Apoptosis by Preventing the Degradation of BIM in Colorectal Cancer.

Colorectal cancer (CRC), a prevalent malignancy worldwide, has prompted extensive research into anticancer drugs. Traditional Chinese medicinal materials offer promising avenues for cancer management due to their diverse pharmacological activities. This study investigated the effects of Notopterygium incisum, a traditional Chinese medicine named Qianghuo (QH), on CRC cells and the underlying mechanism. The sulforhodamine B assay and colony formation assay were employed to assess the effect of QH extract on the proliferation of CRC cell lines HCT116 and Caco-2. Propidium iodide (PI) staining was utilized to detect cell cycle progression, and PE Annexin V staining to detect apoptosis. Western blotting was conducted to examine the levels of apoptotic proteins, including B-cell lymphoma 2-interacting mediator of cell death (BIM), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (BAX) and cleaved caspase-3, as well as BIM stability after treatment with the protein synthesis inhibitor cycloheximide. The expression of BAX was suppressed using lentivirus-mediated shRNA to validate the involvement of the BIM/BAX axis in QH-induced apoptosis. The in vivo effects of QH extract on tumor growth were observed using a xenograft model. Lastly, APCMin+ mice were used to study the effects of QH extract on primary intestinal tumors. QH extract exhibited significant in vitro anti-CRC activities evidenced by the inhibition of cell proliferation, perturbation of cell cycle progression, and induction of apoptosis. Mechanistically, QH extract significantly increased the stability of BIM proteins, which undergo rapid degradation under unstressed conditions. Knockdown of BAX, the downstream effector of BIM, significantly rescued QH-induced apoptosis. Furthermore, the in vitro effect of QH extract was recapitulated in vivo. QH extract significantly inhibited the tumor growth of HCT116 xenografts in nude mice and decreased the number of intestinal polyps in the APCMin+ mice. QH extract promotes the apoptosis of CRC cells by preventing the degradation of BIM.