Rapid Development Of Resistance Research Articles

Extensively Drug-Resistant Klebsiellapneumoniae Counteracts Fitness and Virulence Costs That Accompanied Ceftazidime-Avibactam Resistance Acquisition.

ABSTRACTThe ability of extensively drug-resistant (XDR) Klebsiella pneumoniae to rapidly acquire resistance to novel antibiotics is a global concern. Moreover, Klebsiella clonal lineages that successfully combine resistance and hypervirulence have increasingly occurred during the last years. However, the underlying mechanisms of counteracting fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we investigated whether and how an XDR sequence type (ST)307 K. pneumoniae strain developed resistance against the novel drug combination ceftazidime-avibactam (CAZ-AVI) using experimental evolution. In addition, we performed in vitro and in vivo assays, molecular modeling, and bioinformatics to identify resistance-conferring processes and explore the resulting decrease in fitness and virulence. The subsequent amelioration of the initial costs was also addressed. We demonstrate that distinct mutations of the major nonselective porin OmpK36 caused CAZ-AVI resistance that persists even upon following a second experimental evolution without antibiotic selection pressure and that the Klebsiella strain compensates the resulting fitness and virulence costs. Furthermore, the genomic and transcriptomic analyses suggest the envelope stress response regulator rpoE and associated RpoE-regulated genes as drivers of this compensation. This study verifies the crucial role of OmpK36 in CAZ-AVI resistance and shows the rapid adaptation of a bacterial pathogen to compensate fitness- and virulence-associated resistance costs, which possibly contributes to the emergence of successful clonal lineages.IMPORTANCE Extensively drug-resistant Klebsiella pneumoniae causing major outbreaks and severe infections has become a significant challenge for health care systems worldwide. Rapid resistance development against last-resort therapeutics like ceftazidime-avibactam is a significant driver for the accelerated emergence of such pathogens. Therefore, it is crucial to understand what exactly mediates rapid resistance acquisition and how bacterial pathogens counteract accompanying fitness and virulence costs. By combining bioinformatics with in vitro and in vivo phenotypic approaches, this study revealed the critical role of mutations in a particular porin channel in ceftazidime-avibactam resistance development and a major metabolic regulator for ameliorating fitness and virulence costs. These results highlight underlying mechanisms and contribute to the understanding of factors important for the emergence of successful bacterial pathogens.

Mefloquine induces ER stress and apoptosis in BRAFi-resistant A375-BRAFV600E /NRASQ61K malignant melanoma cells targeting intracranial tumors in a bioluminescent murine model.

Molecularly targeted therapeutics have revolutionized the treatment of BRAFV600E -driven malignant melanoma, but the rapid development of resistance to BRAF kinase inhibitors (BRAFi) presents a significant obstacle. The use of clinical antimalarials for the investigational treatment of malignant melanoma has shown only moderate promise, attributed mostly to inhibition of lysosomal-autophagic adaptations of cancer cells, but identification of specific antimalarials displaying single-agent antimelanoma activity has remained elusive. Here, we have screened a focused library of clinically used artemisinin-combination therapeutic (ACT) antimalarials for the apoptotic elimination of cultured malignant melanoma cell lines, also examining feasibility of overcoming BRAFi-resistance comparing isogenic melanoma cells that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E /NRASQ61 vs. BRAFi-resistant A375-BRAFV600E /NRASQ61K ). Among ACT antimalarials tested, mefloquine (MQ) was the only apoptogenic agent causing melanoma cell death at low micromolar concentrations. Comparative gene expression-array analysis (A375-BRAFV600E /NRASQ61 vs. A375-BRAFV600E /NRASQ61K ) revealed that MQ is a dual inducer of endoplasmic reticulum (ER) and redox stress responses that precede MQ-induced loss of viability. ER-trackerTM DPX fluorescence imaging and electron microscopy indicated ER swelling, accompanied by rapid induction of ER stress signaling (phospho-eIF2α, XBP-1s, ATF4). Fluo-4 AM-fluorescence indicated the occurrence of cytosolic calcium overload observable within seconds of MQ exposure. In a bioluminescent murine model employing intracranial injection of A375-Luc2 (BRAFV600E /NRASQ61K ) cells, an oral MQ regimen efficiently antagonized brain tumor growth. Taken together, these data suggest that the clinical antimalarial MQ may be a valid candidate for drug repurposing aiming at chemotherapeutic elimination of malignant melanoma cells, even if metastasized to the brain and BRAFi-resistant.

Fitness cost of malathion resistance in Egyptian Culex pipiens populations.

The extensive use of insecticides in agriculture and public health has resulted in the rapid development of insecticide resistance in mosquito populations. Therefore, in this study, we aimed to evaluate insecticide resistance costs on the fitness of Culex pipiens. Two Cx. pipiens field populations (Beheira malathion-resistant and Gharbia malathion-susceptible) were compared to the reference (sensitive) population. The biochemical composition and expression of four genes relevant to insecticide resistance were estimated in third instar larvae. Adult survival, female fecundity and egg hatchability were also determined. As per our findings, it was found that the total protein and carbohydrate contents in Beheira malathion-resistant larvae were significantly lower than that in the reference larvae. Beheira malathion-resistant larvae had higher phenoloxidase (PO) specific activity than the reference population. In terms of the relevant genes, only cytochrome P450 (CYP6F1) expression showed elevated levels in the Gharbia malathion-susceptible population compared to the Beheira malathion-resistant population. In esterases (Estα and Estβ) and glutathione S-transferase, the tested populations did not show any significant differences. Compared to the reference mosquito population, Gharbia malathion-susceptible Cx. pipiens males exhibited significantly longer median survival. Female fecundity and hatchability showed nonsignificant differences among the populations tested. In conclusion, malathion resistance can induce lower protein and carbohydrate contents, but higher PO activity in larvae.

Use of metalaxyl against some soil plant pathogens of the class Peronosporomycetes - A review and two case studies

Upon its discovery and implementation in plant protection, metalaxyl became one of the most important fungicides against Phytophthora infestans, but its efficiency has also been proven against other soil pathogens of the class Peronosporomycetes. The most important genus – Phytophthora comprises more than 150 plant pathogens, which cause significant losses in crop production or damage to natural plant associations. Many species of related genera Pythium, Phytopythium and Globisporangium have a similar ability as the species of Phytophthora. Those pathogens are able to quickly spread in wet soils by actively movable zoospores or in the air by means of zoosporangia; they are able to persist in an environment for long periods once they are introduced into the locality, having durability from their resting structures (chlamydospores, hyphal swelling and oospores). Metalaxyl has proven to be very efficient against these pathogens. However, shortly after its release, the rapid development of resistance against this compound was recorded in many species of the class Peronosporomycetes. Such easily developed resistance is due to the monogenic nature of the resistance, which also determines any anti-resistant strategies. The solution of this issue rests in the cautious use of metalaxyl, with consideration given to these strategies, and should be based also on precise information about the environment and the present pathogenic agents.

Overcoming Stenotrophomonas maltophilia Resistance for a More Rational Therapeutic Approach

Stenotrophomonas maltophilia is an underappreciated source of morbidity and mortality among gram-negative pathogens. Effective treatment options with acceptable toxicity profiles are limited. Phenotypic susceptibility testing via commercial automated test systems is problematic and no Food and Drug Administration breakpoints are approved for any of the first-line treatment options for S maltophilia. The lack of modern pharmacokinetic/pharmacodynamic data for many agents impedes dose optimization, and the lack of robust efficacy and safety data limits their clinical utility. Levofloxacin has demonstrated similar efficacy to trimethoprim-sulfamethoxazole, although rapid development of resistance is a concern. Minocycline demonstrates the highest rate of in vitro susceptibility, however, evidence to support its clinical use are scant. Novel agents such as cefiderocol have exhibited promising activity in preclinical investigations, though additional outcomes data are needed to determine its place in therapy for S maltophilia. Combination therapy is often employed despite the dearth of adequate supporting data.

Ras Pathway Activation and MEKi Resistance Scores Predict the Efficiency of MEKi and SRCi Combination to Induce Apoptosis in Colorectal Cancer.

Simple SummaryInhibition of MEK has been proposed as a means to address mutant RAS colorectal cancer (CRC). However, MEK inhibitor adaptive resistance has led to reduced clinical utility of this new drug. Our studies have suggested the potential for the addition of an SRC inhibitor to prevent the development of resistance to MEK inhibitors. Moreover, we have identified that gene expression signature scores for RAS pathway activation, and MEK inhibitor resistance may be useful biomarkers in determining CRC drug sensitivity to the novel combination of Trametinib and Dasatinib.Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. The RAS pathway is activated in more than 55% of CRC and has been targeted for therapeutic intervention with MEK inhibitors. Unfortunately, many patients have de novo resistance, or can develop resistance to this new class of drugs. We have hypothesized that much of this resistance may pass through SRC as a common signal transduction node, and that inhibition of SRC may suppress MEK inhibition resistance mechanisms. CRC tumors of the Consensus Molecular Subtype (CMS) 4, enriched in stem cells, are difficult to successfully treat and have been suggested to evade traditional chemotherapy agents through resistance mechanisms. Here, we evaluate targeting two pathways simultaneously to produce an effective treatment by overcoming resistance. We show that combining Trametinib (MEKi) with Dasatinib (SRCi) provides enhanced cell death in 8 of the 16 tested CRC cell lines compared to treatment with either agent alone. To be able to select sensitive cells, we simultaneously evaluated a validated 18-gene RAS pathway activation signature score along with a 13-gene MEKi resistance signature score, which we hypothesize predict tumor sensitivity to this dual targeted therapy. We found the cell lines that were sensitive to the dual treatment were predominantly CMS4 and had both a high 18-gene and a high 13-gene score, suggesting these cell lines had potential for de novo MEKi sensitivity but were subject to the rapid development of MEKi resistance. The 13-gene score is highly correlated to a score for SRC activation, suggesting resistance is dependent on SRC. Our data show that gene expression signature scores for RAS pathway activation and for MEKi resistance may be useful in determining which CRC tumors will respond to the novel drug combination of MEKi and SRCi.

Larvicidal Activities and Synergistic Effects of Essential Oils against Anopheles funestus and Culex quinquefasciatus (Diptera: Culicidae) from Kisumu, Kenya

Rapid development of resistance in vector mosquitoes to synthetic insecticides is a major challenge for malaria control. The use of plant-derived essential oils (EOs) is an attractive strategy in controlling mosquito populations because they are environmentally safe and may have a lower chance of developing resistance. This study assessed the larvicidal activities of EOs from Lantana camara, Lippia multiflora, Lippia chevalieri, and Cymbopogon schoenanthus against Anopheles funestus and Culex quinquefasciatus. The 3rd–4th instar larvae were tested using a World Health Organization (WHO)-modified protocol to evaluate larval mortality 24 h after exposure to EOs and their binary combinations. Culex quinquefasciatus larvae were more susceptible to EOs than An. funestus larvae. For Cx. quinquefasciatus, the lethal concentrations at 50% mortality (LC50s) of EOs from C. schoenanthus, L. multiflora, L. camara, and L. chevalieri were 23.32, 27.24, 38.54, and 54.11 ppm, respectively; whereas for An. funestus, the EO LC50s were 120.5, 67.5, 49.21, and 105.74 ppm, respectively. Synergistic effects were observed using EOs from C. schoenanthus + L. multiflora (LC50 = 44.05 ppm) on An. funestus, while L. camara + L. chevalieri (LC50 = 33.16 ppm), L. chevalieri + C. schoenanthus (LC50 = 12.08 ppm), and L. multiflora + L. chevalieri (LC50 = 20.61 ppm) were synergistic for Cx. quinquefasciatus. These results indicate the potential of EOs derived from local plants and their binary combinations as botanical larvicides. The EOs could be used as future ecofriendly agents to control these vectors.

Spinetoram resistance drives interspecific competition between Megalurothrips usitatus and Frankliniella intonsa.

Species displacement by the outcome of interspecific competition is of particular importance to pest management. Over the past decade, spinetoram has been extensively applied in control of the two closely related thrips Megalurothrips usitatus and Frankliniella intonsa worldwide, while whether its resistance is implicated in mediating interspecific interplay of the two thrips remains elusive to date. Field population dynamics (from 2017 to 2019) demonstrated a trend toward displacement of F. intonsa by M. usitatus on cowpea crops, supporting an existing interspecific competition. Following exposure to spinetoram, M. usitatus became the predominate species, which suggests the use of spinetoram appears to be responsible for mediating interspecific interactions of the two thrips. Further annual and seasonal analysis (from 2016 to 2020) of field-evolved resistance dynamics revealed that M. usitatus developed remarkably higher resistance to spinetoram compared to that of F. intonsa, implying a close relationship between evolution of spinetoram resistance and their competitive interactions. After 12 generations of laboratory selection, resistance to spinetoram in M. usitatus and F. intonsa increased up to 64.50-fold and 28.33-fold, and the average realized heritability (h2 ) of resistance was calculated as 0.2550 and 0.1602, respectively. Interestingly, two-sex life table analysis showed that the spinetoram-resistant strain of F. intonsa exhibited existing fitness costs, but not the M. usitatus. These indicate that a rapid development of spinetoram resistance and the lack of associated fitness costs may be the mechanism underlying recent dominance of M. usitatus over F. intonsa. Collectively, our results uncover the involvement of insecticide resistance in conferring displacement mechanism behind interspecific competition, providing a framework for understanding the significance of the evolutionary relationships among insects under ongoing changing environments. These findings also can be invaluable in proposing the most appropriate strategies for sustainable thrips control programs. © 2022 Society of Chemical Industry.

Campylobacter jejuni Developed the Resistance to Bacteriophage CP39 by Phase Variable Expression of 06875 Encoding the CGPTase.

Bacteriophage (phage) is regarded as an antimicrobial alternative for Campylobacter in food production. However, the development of phage resistance to the host is a main concern for the phage application. This study characterized the phage CP39 and investigated the phage resistance of CP39 in Campylobacter jejuni NCTC12662. We determined that phage CP39 belonged to the Myoviridae family by the WGS and phylogenetic analysis. Phage CP39 was confirmed as a capsular polysaccharide (CPS)-dependent phage by primary C. jejuni phage typing. It was further confirmed that the phage could not be adsorbed by the acapsular mutant ΔkpsM but showed the same lytic ability in both the wild-type strain NCTC 12662 and the ΔmotA mutant lacking motile flagella filaments. We further determined that the 06875 gene encoding CDP-glycerol:poly (glycerophosphate) glycerophosphotransferase (CGPTase) in the CPS loci was related to phage CP39 adsorption by SNP analysis and observed a rapid development of phage resistance in NCTC 12662 during the phage infection. Furthermore, we observed a high mutation frequency of 06875 (32%), which randomly occurred in nine different sites in the gene according to colony PCR sequencing. The mutation of the 06875 gene could cause the phase variable expression of non-functional protein and allow the bacteria against the phage infection by modifying the CPS. Our study confirmed the 06875 gene responsible for the CPS-phage adsorption for the first time and demonstrated the phase variable expression as a main mechanism for the bacteria to defend phage CP39. Our study provided knowledge for the evolutionary adaption of bacteria against the bacteriophage, which could add more information to understand the phage resistance mechanism before applying in the industry.

A genome‐wide CRISPR activation screen reveals Hexokinase 1 as a critical factor in promoting resistance to multi‐kinase inhibitors in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine-kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second-line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib-resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success.

Toxicological characteristic and bactericidal properties of Keggin-type heteropolyacids

Introduction. In modern reality the effectiveness of disinfection measures is often complicated by rapid development of microbial resistance to known disinfectants, therefore a constant search for new effective, safe disinfectants and compositions is necessary. Polyoxometallates (POMs), especially Keggin’s heteropolyacids (HPAs), are a promising class of inorganic compounds with a broad biological activity, including antiviral and antibacterial ones. So the research of HPAs toxicity and bactericidal activity for the creation of new disinfectants or optimization of known disinfectants by combining with HPAs are essential and actual. Materials and methods. HPAs samples H3PW12O40, H3PMo12O40, H4SiW12O40, H4SiMo12O40, H4PMo11VO40, H5PMo10V2O40, H4PW11VO40 were evaluated by the cytotoxicity index (IC50) in three cell lines by the MTT method using MTT-reagent 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. HPAs acute toxicity in white mice (administration into the stomach and intraperitoneally) was tested in accordance with GOST 12.1.007-76. Bactericidal activity of HPAs and compositions with hydrogen peroxide was determined against test microorganisms S. aureus and E. coli by the suspension method. The results were supplemented by electron microscopic examination. Results. Vanadium-containing HPAs structures had higher cytotoxicity. All HPAs corresponded to hazard class III in terms of acute toxicity. Bactericidal activity of HPAs and HPAs combinations with hydrogen peroxide was revealed. The incorporation of two vanadium atoms into HPAs structure increased the bactericidal activity. Electron microscopic examination showed more pronounced morphological changes in the ultrastructure of E.coli treated with HPAs / hydrogen peroxide combinations. Mechanism of the bactericidal activity may be associated with HPAs penetration through a damaged membrane of porin channels of bacterial cells. Explanations are also given for another possible mechanism of HPAs action. Limitations. The study is limited to researching toxicological characteristics and bactericidal properties of HPAs, their combinations with hydrogen peroxide. The limited number of HPAs and disinfectants samples is associated with the complexity of the biological experiments. The choice of three cell lines is determined by three different sources of their origin. The suspension method for assessing the antimicrobial activity of substances was performed under the relevant GOST. Test cultures of S. aureus and E. coli were used, other pathogens were not considered. The experiments in vivo were carried out under guidelines for the protection of experimental animals, and their limited number is due to the cost of animals and current ethical views on experiments in vivo. Conclusion. It has been shown that prospects of both HPAs research and combining HPAs with disinfectants having a different mechanism of action in order to enhance a disinfecting effect and reduce the risk of developing microbial resistance to disinfectants.

Comparative analysis of resistance to ALS-inhibiting herbicides in smallflower umbrella sedge (Cyperus difformis) populations from direct-seeded and puddled-transplanted rice systems

AbstractThe transition from puddled-transplanted rice (Oryza sativa L.) (PTR) to direct-seeded rice (DSR) is gaining popularity in central China. In contrast, the PTR system is the most common practice in southwest China. Weeds are a major problem in the paddy fields of the DSR systems, and herbicides are widely used for weed control. However, the increased frequency and rate of herbicide use leads to the rapid evolution of resistance. Smallflower umbrella sedge (Cyperus difformis L.) is a troublesome weed species in rice fields of China and is usually controlled by the acetolactate synthase (ALS)-inhibiting herbicide bensulfuron-methyl. Here, we collected 32 C. difformis populations from DSR systems (Hunan Province) and PTR systems (Guangxi Province) and investigated their resistance to bensulfuron-methyl. Results revealed 80% (8 out of 10) populations from Hunan Province and 14% (3 out of 22) populations from Guangxi Province had evolved resistance to bensulfuron-methyl. Five populations from Hunan Province (HN-2, HN-3, HN-5, HN-9, HN-10) possessing the Trp-574-Leu mutation had high-level resistance (ranging from 169- to >1,309-fold) based on GR50 ratios. The resistant populations from Guangxi Province had a lower level of resistance to bensulfuron-methyl due to a Pro-197-Ser mutation. The Asp-376-Glu mutation was only identified in the HN-4 population. In addition, the GX-3 population from the PTR systems was resistant to bensulfuron-methyl without ALS gene mutations, indicating non–target site resistance (NTSR). Although some resistant populations of both regions exhibited cross-resistance to multiple ALS-inhibiting herbicides, including pyrazosulfuron-ethyl, bispyribac-sodium, penoxsulam, and imazapic, sensitivity was also detected to the auxin herbicide MCPA and the photosystem II–inhibiting herbicides bentazone and propanil. These results indicate that cultivation practices affect resistance evolution in C. difformis. DSR systems exert high selection pressure by selecting the Trp-574-Leu mutation, resulting in high-level resistance. In contrast, a mutation at Pro-197 plus NTSR likely plays a significant role in ALS resistance in the PTR systems.

Enhanced CXCL12/CXCR4 signaling increases tumor progression in radiation‑resistant pancreatic cancer.

Pancreatic cancer (PaCa) exhibits one of the poorest prognoses among all gastrointestinal cancers due to the rapid development of treatment resistance, which renders chemotherapy and radiotherapy no longer effective. However, the mechanisms through which PaCa becomes resistant to radiotherapy are unknown. Here, we established radiation-resistant PaCa cell lines to investigate the factors involved in radiation resistance. The role of the C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis in radiation resistance in PaCa and the effects of a CXCR4 antagonist on radiation-resistant PaCa cell lines were investigated. As confirmed by immunofluorescence staining, reverse transcription quantitative polymerase chain reaction, and western blotting, the expression of CXCR4 was higher in radiation-resistant PaCa cell lines than that noted in normal PaCa cell lines. The invasion ability of radiation-resistant PaCa cell lines was greater than that of normal cell lines and was enhanced by CXCL12 treatment and coculture with fibroblasts; this enhanced invasion ability was suppressed by the CXCR4 antagonist AMD070. Irradiation after treatment with the CXCR4 antagonist suppressed the colonization of radiation-resistant PaCa cell lines. In conclusion, the CXCL12/CXCR4 axis may be involved in the radiation resistance of PaCa. These findings may facilitate the development of novel treatments for PaCa.

LncRNAs LCETRL3 and LCETRL4 at chromosome 4q12 diminish EGFR-TKIs efficiency in NSCLC through stabilizing TDP43 and EIF2S1

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are effective targeted therapy drugs for advanced non-small cell lung cancer (NSCLC) patients carrying sensitized EGFR mutations. The rapid development of EGFR-TKIs resistance represents a major clinical challenge for managing NSCLC. The chromosome 4q12 is the first genome-wide association study (GWAS)-reported locus associated with progression-free survival (PFS) of NSCLC patients treated with EGFR-TKIs. However, the biological significance of the noncoding transcripts at 4q12 in NSCLC remains elusive. In the present study, we identified two 4q12 long noncoding RNAs (lncRNAs) LCETRL3 and LCETRL4 which could significantly dimmish EGFR-TKIs efficiency. In line with their oncogenic role, evidently higher LCETRL3 and LCETRL4 levels were observed in NSCLC tissues as compared with normal specimens. Importantly, lncRNA LCETRL3 can interact with oncoprotein TDP43 and inhibit ubiquitination and degradation of TDP43. Similarly, lncRNA LCETRL4 can bind and stabilize oncoprotein EIF2S1 through reducing ubiquitin-proteasome degradation of EIF2S1. In particular, elevated levels of LCETRL3 or LCETRL4 in NSCLC cells resulted in stabilization of TDP43 or EIF2S1, increased levels of NOTCH1 or phosphorylated PDK1, activated AKT signaling and, thus, EGFR-TKIs resistance. Taken together, our data revealed a novel model that integrates two lncRNAs transcribed from the 4q12 locus into the regulation of EGFR-TKIs resistance in NSCLC. These findings shed new light on the importance of functionally annotating lncRNAs in the GWAS loci and provided insights to declare novel druggable targets, i.e., lncRNAs, which may unlock the therapeutic potential of EGFR-TKIs resistant NSCLC in the clinic.

Targeting KRAS mutant lung cancer: light at the end of the tunnel.

For decades, KRAS mutant lung adenocarcinomas (LUAD) have been refractory to therapeutic strategies based on personalized medicine owing to the complexity of designing inhibitors to selectively target KRAS and downstream targets with acceptable toxicities. The recent development of selective KRASG12C inhibitors represents a landmark after 40 years of intense research efforts since the identification of KRAS as a human oncogene. Here, we discuss the mechanisms responsible for the rapid development of resistance to these inhibitors, as well as potential strategies to overcome this limitation. Other therapeutic strategies aimed at inhibiting KRAS oncogenic signaling by targeting either upstream activators or downstream effectors are also reviewed. Finally, we discuss the effect of targeting the mitogen‐activated protein kinase (MAPK) pathway, both based on the failure of MEK and ERK inhibitors in clinical trials, as well as on the recent identification of RAF1 as a potential target due to its MAPK‐independent activity. These new developments, taken together, are likely to open new avenues to effectively treat KRAS mutant LUAD.

Ингибитор АКТ-сигнального пути потенцирует цитотоксическую активность доксорубицина в отношении клеток остеосарком in vitro

Activation of receptor and nonreceptor tyrosyne kinases (TK) is detected in a broad spectrum of human malignancies, including soft tissue sarcomas (STS) and osteosarcomas (OS) and in some cases correlates with poor prognosis and clinical outcomes. Despite the topoisomerase II inhibitor doxorubicin is broadly used for treatment of STS and OC as mono- and as a component of the combined chemotherapeutic regimens, rapid development of tumor resistance to doxorubicin is considered to be most significant factor of poor prognosis and bad clinical outcome, especially for the patients with metastatic and reccurent forms of disease. This, in turn, remains a driving force to develop novel effective approaches to increase sensitivity of tumor cells to this chemotherapeutic agent and overcome drug resistance. Our current data illustates activation of c-Met-, FGFR-, AKT-, и МAPK-kinases in OS cells, whereas inhibition of their activities with corresponding TK inhibitors (TKIs) (crizotinib, BGJ398, MK-2206, and U0126, respectively) enhances cytotoxic activity of doxorubicin against OS cells. The most prominent synergism between doxorubicin and TKIs inhibitors was found for AKT inhibitor MK- 2206. We found that MK-2206 significantly enhanced sensitivity of OS cells to doxorubicin and induced their apoptosis. Thus, activation of AKT signaling in OS might be an attractive therapeutic target to increase OS sensitivy to chemotherapeutic agents, in particular, doxorubicin. This in turn provide the molecular basis for the effective further use of the combined (AKT- and topoisomerase II inhibitors) therapy for patinets with OS.

Integrating a covalent probe with ubiquicidin fragment enables effective bacterial infection imaging.

Developing potent and novel bacterial imaging agents remains formidable due to the rapid development of bacterial resistance. Ubiquicidin and its derivatives are the most studied antimicrobial peptides that bind to anionic membranes of a broad range of bacterial pathogens. Studies reveal that UBI (29-41) labeled with 99mTc and 68Ga could distinguish sterile inflammation from infection. A significant challenge that remains for cationic peptides is their poor salt tolerance. The present study deliberates the increment of UBI (29-41) peptide interaction with the bacterial membrane by incorporating 2-acetylphenylboronic acid (2-APBA) as a covalent probe and developing infection imaging probes with improved retention at the target. Given that both 99mTc-UBI (29-41) and 99mTc-UBI (29-41)-2-APBA peptide complexes are stable in serum over 16 h, 99mTc-UBI (29-41)-2-APBA shows enhanced uptake in S. aureus cells as compared to 99mTc-UBI (29-41). SPECT imaging in a mouse model of infection exhibited a higher target to non-target ratio after 2 h in the case of 99mTc-UBI (29-41)-2-APBA. The present study reveals a synergistic mechanism of target binding through covalent conjugation and non-covalent interaction, which could be a potential strategy for improving bacterial infection imaging. As a proof of concept, 99mTc-UBI (29-41)-2-APBA elicits our hypothesis by in vivo imaging of bacterial infection.

A phage cocktail in controlling phage resistance development in multidrug resistant Aeromonas hydrophila with great therapeutic potential

Aeromonas hydrophila (A. hydrophila) is an opportunistic pathogen of fish-human-livestock, which poses a threat to the development of aquaculture. Lytic phage has long been considered as an effective bactericidal agent. However, the rapid development of phage resistance seriously hinders the continuous application of lytic phages. In our study, a new bacteriophage vB_ AhaP_PZL-Ah8 was isolated from sewage and its characteristics and genome were investigated. Phage vB_ AhaP_PZL-Ah8 has been classified as the member of the Podoviridae family, which exhibited the latent period was about 30min. As revealed from the genomic sequence analysis, vB_ AhaP_PZL-Ah8 covered a double-stranded genome of 40,855 bp (exhibiting 51.89% G+C content), with encoding 52 predicted open reading frames (ORFs). The results suggested that the combination of vB_ AhaP_PZL-Ah8 and another A. hydrophila phage vB_ AhaP_PZL-Ah1 could improve the therapeutic efficacy both in vitro and in vivo. The resistance mutation frequency of A. hydrophila cells infected with the mixture phage (vB_ AhaP_PZL-Ah8+ vB_ AhaP_PZL-Ah1) was significantly lower than cells treated with single phage (P <0.01). Phage therapy in vivo showed that the survival rate in the mixture phage treatment group was significantly higher than that in single phage treatment group.

Nelfinavir Induces Cytotoxicity towards High-Grade Serous Ovarian Cancer Cells, Involving Induction of the Unfolded Protein Response, Modulation of Protein Synthesis, DNA Damage, Lysosomal Impairment, and Potentiation of Toxicity Caused by Proteasome Inhibition.

Simple SummaryHigh-grade serous ovarian cancer (HGSOC) accounts for 70% of all ovarian-cancer-related deaths. Mainstay treatment with platinum-based drugs following surgery results in favorable outcomes in the majority of patients; however, in >80% of cases, the disease relapses with eventual drug resistance. As such, urgent development of improved alternative therapies is necessary for HGSOC patients with lower life expectancy. Rapid repurposing of market available drugs for cancer therapy is a cost-effective alternative to bypass the decade-long traditional drug development pipeline. Among potential drug-repurposing candidates, nelfinavir (NFV)—an anti-infective agent to treat acquired immunodeficiency syndrome (AIDS)—has shown anti-cancer effects against diverse cancers; however, its remedial benefits against HGSOC are unknown. In this study, we explored how NFV targets HGSOC cells obtained from patients at platinum-sensitive and -resistant stages. We observed beneficial efficacy elicited by NFV against HGSOC in both disease conditions through multiple mechanistic avenues, suggesting positive drug-repurposing prospects.High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction in the HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction in clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. In addition, dose- and time-dependent phosphorylation of H2AX indicated NFV-mediated DNA damage, which was associated with decreased survival and proliferation signals driven by the AKT and ERK pathways. NFV also mediated a dose-dependent increase in endoplasmic reticulum stress-related molecules associated with long-term inhibition of protein synthesis and concurrent cell death; such events were accompanied by a proapoptotic environment, signaled by increased phospho-eIF2α, ATF4, and CHOP, increased Bax/Bcl-2 ratio, and cleaved executer caspase-7. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile.

Effect of Cry Toxins on Xylotrechus arvicola (Coleoptera: Cerambycidae) Larvae

Simple SummaryXylotrechus arvicola is a destructive pest in vineyards (Vitis vinifera) in the main wine-producing areas of the Iberian Peninsula. X. arvicola larvae bore into the grapevine wood-making galleries, thus damaging the plant both directly and indirectly. The susceptibility of X. arvicola larvae to five coleopteran toxic Cry proteins was evaluated under laboratory conditions in order to deepen the knowledge of the effect of these proteins on this insect throughout its biological development. The Cry proteins tested could be applied to control X. arvicola larvae since they were able to kill them and cause serious alterations in the larvae during the remaining months of development that followed. The data presented suggest that these Cry proteins can be used as bioinsecticides against the larvae of this insect, in order to avoid the rapid evolution of resistance against these toxins since not all of the larvae were killed and thus increase vine wood protection.The beetle Xylotrechus arvicola is a destructive pest in vineyards (Vitis vinifera) in the main wine-producing areas of the Iberian Peninsula. X. arvicola larvae bore into the grapevine wood-making galleries, thus damaging the plant both directly and indirectly; the latter through the proliferation of wood fungi, which can invade the inside of the plant, decreasing the quality and quantity of its production. The susceptibility of X. arvicola larvae to five coleopteran toxic Cry proteins (Cry1B, Cry1I, Cry3A, Cry7A, and Cry23/37) was evaluated under laboratory conditions in order to deepen the knowledge of the effect of these proteins on this insect throughout its biological development. Cry7Ab and Cry1Ba were the most effective in controlling X. arvicola larvae due to the significant reduction in larvae survival (32.9 and 25.9 days, respectively), and by causing serious alterations in the larvae during the remaining months of their development. The developmental stage of the prepupal and pupal stages was not affected by the previous ingestion of Cry proteins. The Cry proteins tested could be applied to control X. arvicola larvae since they were able to kill them and cause serious alterations in the larvae during the remaining months of development that followed. The data presented suggest that these Cry proteins can be used as bioinsecticides against the larvae of this insect, applying them only at the moment when the larvae hatch from the egg outside the grapevine wood (this would only be useful and justified if the economic threshold is exceeded) in order to avoid the rapid evolution of resistance against these toxins since not all of the larvae were killed and thus increase vine wood protection.