Search

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.anbehav.2008.08.020 Byline: M. Aluja, D. Perez-Staples, J. Sivinski, A. Sanchez, J. PiA[+ or -]ero Abstract: We investigated the effects of male size and adult diet on male sexual competitiveness and lifetime reproductive success of female mates in two tephritid flies with contrasting natural histories, the Mexican fruit fly, Anastrepha ludens, and the guava fruit fly, A. striata. Small, medium and large males received either a low-quality diet (sucrose offered every third day) or a high-quality diet (sucrose and protein offered ad libitum). Regardless of size or species, males on the high-quality diet copulated significantly more often across the 4 observation days than males on the low-quality diet. For A. ludens, both size and diet influenced territory defence by resident males, but only size affected the likelihood of success by invading males. Females that copulated with a low-quality fed male had significantly shorter maximum longevity, although there was no effect of male diet on the proportion of eclosed eggs. For A. striata, diet, but not size, influenced territory defence by invading males. Females discriminated strongly against males fed a low-quality diet but not against smaller males. However, females that copulated with medium and small males had lower fecundities than those copulating with large males. We discuss the costs for females of mating with smaller males and their inability to distinguish between males of different conditions, as well as interspecific differences in mate choice and postcopulatory consequences for females in terms of the ecological differences that distinguish these two species. Author Affiliation: (a) Instituto de Ecologia, A.C., Xalapa, Veracruz, Mexico (a ) INBIOTECA, Universidad Veracruzana, Xalapa, Veracruz, Mexico (a ) Center for Medical, Agricultural & Veterinary Entomology, Gainesville, FL, U.S.A. Article History: Received 18 January 2008; Revised 18 February 2008; Accepted 6 August 2008 Article Note: (miscellaneous) MS. number: A08-00043

Protein, lipid, carbohydrate, and energy contents of three artificial diets (Xal2, Met1, and Met2) used for laboratory-rearing and mass-rearing the Mexican fruit fly, Anastrepha ludens (Loew), for a sterile insect technique program were measured. The larval survival, pupation, pupal weight, adult emergence, sex ratio, and flight capacity of the flies reared on each of these diets were also quantified. The diet with the highest nutrient and energy content was Xal2 followed by Met2 and Met1, but larval recovery and percent pupation was significantly higher in flies reared on either the Met1 or Met2 diets. A. ludens reared on Xal2 exhibited the highest proportion of adults capable of flight. No other response variable differed significantly among the three diets tested. This suggests that a high content of nutrients and multiple sources of protein (dried yeast and wheat germ in the case of the Xal2 diet) do not necessarily improve overall performance or fly quality. We conclude that nutritious diets for A. ludens can be modified to reduce their cost without compromising the performance of artificially reared flies. [ABSTRACT FROM PUBLISHER]

The success of the sterile insect technique (SIT) requires the release of males that can compete with wild males. Elevated juvenile hormone (JH) levels and hydrolysed yeast-enriched adult diets improve the sexual attractiveness and competitiveness of sterile male Anastrepha ludens (Loew; Mexican fruit fly), used in large-scale SIT programmes in Mexico and Texas. We applied methoprene (a JH analogue) to A. ludens using two methods, topically on individual sterile adults and mass- immersion of pupae after irradiation. Hydrolysed yeast was either used dry or in agar blocks. Laboratory and field cage experiments were conducted to compare male maturation and sexual performance when males exposed to four different treatments competed for females: (M+P+) application of methoprene and sugar plus hydrolysed yeast as adult food; (M+P−) application of methoprene and sugar only as adult food; (M−P+) no application of methoprene and sugar plus hydrolysed yeast as adult food; and (M−P−) no application of methoprene and sugar only as adult food. Methoprene and hydrolysed yeast accelerated the male sexual maturation from 9 to 5 days after the emergence. Combined application of methoprene and hydrolysed yeast had a significant effect on increased male sexual performance in both laboratory and field cage tests. Methoprene application also accelerates female maturation, although less than in males. The substantial improvements in male sexual performance and sexual maturation that can be obtained by incorporating the use of JH analogue and hydrolysed protein, and the development of techniques for their practical application on a large scale, can contribute to a more cost-effective deployment of sterile male Mexican fruit flies in SIT programmes. [ABSTRACT FROM AUTHOR]

Abstract: A recently discovered Mexican parasitoid species of Eurytomidae (Hymenoptera), Eurytoma sivinskii Gates and Grissell, has the unique behavior, for its family, of attacking tephritid fruit fly pupae (Anastrepha spp.) on or in the soil. Adults burrowed but did so rarely, thus pupae on the soil surface were significantly more vulnerable than those underground. Females facultatively hyperparasitized other larval-prepupal and pupal parasitoids such as Opius hirtus (Braconidae), Coptera haywardi (Diapriidae) and Pachycrepoideus vindemiae (Pteromalidae). While E. sivinskii developed in the pupae of various other Anastrepha, including, A. serpentina and A. striata, it also attacked cyclorraphous Diptera such as Musca domestica and a tachinid species. The number of expected female offspring (Ro ) was 44.3 when measured as eclosed eggs (i.e., that became larvae) and 34.3 when measured as the number of emerged adults, and the intrinsic rate of natural increase (rm ) was 0.34. This is high relative to other fruit fly parasitoids and suggests that E. sivinskii could rapidly exploit a clumped resource. We conclude that the marginal ability of E. sivinskii to attack buried pupae and the environmental risks it poses through its broad host range and capacity for hyperparasitism make it a poor candidate for tephritid biological control. [Copyright &y& Elsevier]

Field studies in citrus were conducted to compare the following as attractants for the Caribbean fruit fly, Anastrepha suspensa (Loew): torula yeast-borax; propylene glycol (10%); a two-component lure consisting of ammonium acetate and putrescine; a two-component lure consisting of ammonium bicarbonate and putrescine; and a three-component lure consisting of ammonium bicarbonate, methylamine hydrochloride, and putrescine. Various combinations of these attractants in glass McPhail, plastic McPhail-type (Multi-Lure), and sticky panel traps were investigated in two replicated studies. In one study on wild flies, the most effective and least complex trap-lure combination tested was the Multi-Lure with propylene glycol baited with ammonium acetate and putrescine. This trap-lure combination captured significantly more female and male flies than the standard glass McPhail baited with torula yeast-borax in water. All of the trap-lure combinations were female biased, with an overall average of 80.8% (SEM 1.4) flies captured being female. A second study on laboratory-reared, irradiated flies indicated no significant differences among these trap-lure combinations with respect to number of flies recaptured, although rankings based on mean number of flies recovered per trap per day supported results of the first study. The percentage of flies recaptured that were female (83.0%, SEM 0.9) was statistically the same as in the first study. Weekly percentage recovery of flies during the second study was low, possibly due to our fly release strategy. Future release/recovery studies with laboratory-reared flies would benefit from some basic research on release strategies by using different trap densities and on relating recapture rates of laboratory-reared flies (nonsterile and sterile) to capture rates of wild flies. [ABSTRACT FROM AUTHOR]

Determines the effects of host fruit volatiles on parasitoid host-searching behavior of Diachasmimorpha longicaudata. Effect of host fruit on parasitoid host location; Effect of host fruit and fly species on the performance of parasitoids; Sex ratio of progeny; Relationship between size and longevity and fecundity and sex ratio of progeny.

Two species of Braconidae, Diachasmimorpha longicaudata (Ashmead) and Doryctobracon areolatus (Szepligeti), commonly attack the Caribbeanfruit fly, Anastrepha suspense (Loew), in central Florida. There aretemporal changes in the relative abundance of the 2 parasitoids, with a tendency for D. longicaudata to become more common as the fruiting periods of various host trees progress. An exception occurs when D. longicaudata declines relative to D. areolatm during the late-autumn and winter fruiting of the citrus fruit calamundin, chi Citrofortuella mitis J. Ingram & H. E Moore. During the year there is a spring rise and then an autumn decline in the relative abundance of D. longicaudata. This pattern is not explained by any within-tree changes in fruit size or host larval densities. Many changes in relative abundance are consistent with D. areolatus being superior to D. longicaudata in finding host patches, but inferior at exploiting hosts (counter-balanced competition), Seasonal changes in maximum temperatures are also correlated to changes in the relative abundance of D. longicaudatato D. areolatus. It appears that the outcome of the competition between the 2 species is influenced by climatic conditions. [ABSTRACT FROM AUTHOR]

Abstract: Recent recognition of widespread polyandry in insects has generated considerable interest in understanding why females mate multiple times and in identifying factors that affect mating rate and inhibit female remating. However, little attention has been paid to understanding the question from both a female and male perspective, particularly with respect to factors that may simultaneously influence female remating rates. Here, we report on a study aimed at ascertaining the possible interactive effects that male and female size and diet, and female access to a host could have on mating latency, probability, and duration and female refractory period using two tropical fruit fly species with contrasting life histories. Of all factors tested, adult diet played the most significant role. Both Anastrepha ludens and Anastrepha obliqua males which had constant access to protein and sucrose mated more often, had shorter copulations and induced longer refractory periods in females than males fed a low quality diet (sucrose offered every third day). Female size and the interaction with male diet determined how quickly female A. ludens mated for the first time. Smaller females mated sooner with low quality fed males than with high quality fed males while there was no difference for large females, suggesting that male choice may be at play if high quality fed males discriminate against smaller females. Copulation duration also depended on both male and female nutritional condition, and the interaction between male diet and female size and diet. Large and high quality fed females had shorter copulations regardless of male condition. Importantly, for A. ludens, female refractory period depended on male size and the nutritional condition of both males and females, which could indicate that for this species, female receptivity does not depend only on the condition of the male ejaculate. For A. obliqua refractory period was associated with the interaction between male size and diet and male diet and host presence. We discuss our results in terms of male ability to inhibit female remating and the relative contribution of female condition to this behavior. We also address the importance of studying effects simultaneously on species with contrasting life histories. [Copyright &y& Elsevier]

We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis -sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium . A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.

Molecular responses to alcohol consumption are dynamic, context-dependent, and arise from a complex interplay of biological and external factors. While many have studied genetic risk associated with drinking patterns, comprehensive studies identifying dynamic responses to pharmacologic and psychological/placebo effects underlying binge drinking are lacking. We investigated transcriptome-wide response to binge, medium, and placebo alcohol consumption by 17 healthy heavy social drinkers enrolled in a controlled, in-house, longitudinal study of up to 12 days. Using RNA-seq, we identified 251 and 13 differentially expressed genes (DEGs) in response to binge drinking and placebo, respectively. Eleven protein-coding DEGs had very large effect sizes in response to binge drinking (Cohen's d > 1). Furthermore, binge dose significantly impacted the Cytokine-cytokine receptor interaction pathway (KEGG: hsa04060) across all experimental sequences. Placebo also impacted hsa04060, but only when administered following regular alcohol drinking sessions. Similarly, medium-dose and placebo commonly impacted KEGG pathways of Systemic lupus erythematosus, Neutrophil extracellular trap formation, and Alcoholism based on the sequence of drinking sessions. These findings together indicate the "dose-extending effects" of placebo at a molecular level. Furthermore, besides supporting alcohol dose-specific molecular changes, results suggest that the placebo effects may induce molecular responses within the same pathways regulated by alcohol., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras are not due to loss of DnaJ binding. Although we do not exclude JDPs as additional specificity factors, our data demonstrate substrate specificity among the Hsp70s, which has important implications for inhibitor development in cancer and neurodegeneration.

A pulldown using a biotinylated natural product of interest in the 17β-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), identified the bromodomain and extra-terminal domain (BET) family of proteins (BRD2, BRD3, and BRD4), readers of acetyl-lysine modifications and regulators of gene transcription, as potential cellular targets. BROMOscan bromodomain profiling and biochemical assays support PCC as a BET inhibitor with increased selectivity for bromodomain (BD)-1 of BRD3 and BRD4, and X-ray crystallography and NMR studies uncovered specific contacts that underlie the potency and selectivity of PCC toward BRD3-BD1 over BRD3-BD2. PCC also displays characteristics of a molecular glue, facilitating proteasome-mediated degradation of BRD3 and BRD4. Finally, PCC is more potent than other withanolide analogues and gold-standard pan-BET inhibitor (+)-JQ1 in cytotoxicity assays across five prostate cancer (PC) cell lines regardless of androgen receptor (AR)-signaling status.

A recent study illustrated that a fluorescence polarization assay can be used to identify substrate-competitive Hsp70 inhibitors that can be isoform-selective. Herein, we use that assay in a moderate-throughput screen and report the discovery of a druglike amino-acid-based inhibitor with reasonable specificity for the endoplasmic reticular Hsp70, Grp78. Using traditional medicinal chemistry approaches, the potency and selectivity were further optimized through structure-activity relationship (SAR) studies in parallel assays for six of the human Hsp70 isoforms. The top compounds were all tested against a panel of cancer cell lines and disappointingly showed little effect. The top-performing compound, 8 , was retested using a series of endoplasmic reticulum (ER) stress-inducing agents and found to synergize with these agents. Finally, 8 was tested in a spheroid tumor model and found to be more potent than in two-dimensional models. The optimized Grp78 inhibitors are the first reported isoform-selective small-molecule-competitive inhibitors of an Hsp70-substrate interaction.

While many studies have established the importance of protein homeostasis in tumor progression, little effort has been made to examine the therapeutic potential of targeting the HSP60 chaperonin system. In healthy cells, HSP60 is localized to the mitochondrial matrix; however, emerging evidence indicates HSP60 can be over-expressed and mis-localized to the cytosol of cancer cells, which is hypothesized to promote tumor cell survival and proliferation. This opens a potential avenue to selectively target the aberrant HSP60 in the cytosol as a chemotherapeutic strategy. In the present work, we examined a series of bis-aryl-α,β-unsaturated ketone (ABK) HSP60 inhibitors for their ability to selectively target cancerous vs non-cancerous colon and intestine cells. We found that lead analogs inhibited migration and clonogenicity of cancer cells, with cytotoxicity correlating with the level of aberrant HSP60 in the cytosol., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare the following competing financial interest(s): S.M.J. and E.C. are co-founders of BioEL Inc, which was formed to commercialize GroEL/ES inhibitors, stemming from research conducted in their laboratories, for antibacterial applications., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies., (© 2022 Federation of American Societies for Experimental Biology.)

Background: The Society of Interdisciplinary Placebo Studies (SIPS) was one of many organizations that hosted a virtual scientific conference in response to the COVID-19 pandemic restrictions. Retaining essential benefits of an in-person conference experience was a primary objective for the SIPS conference planning committee and guided the selection of a virtual platform on which to host the 2021 meeting. This article reports on the methods used to design and analyze an engaging, virtual scientific conference, along with the findings and implications for future meetings., Methods: Participant use of and interaction with different features of the conference platform were recorded and exported for analysis. Additionally, all SIPS conference attendees were invited to complete a brief, online post-conference survey that inquired about their perceptions of the SIPS conference specifically as well as their opinions of virtual and hybrid conferences in general. Using these data, we assessed (1) attendance patterns, (2) level of engagement, and (3) attendee satisfaction., Results: The platform recorded 438 unique, active conference attendees who used either a mobile app, web browser, or both to participate during the 3-day program. Seventy-four percent ( N = 324) of active users attended all 3 days with 30 and 26 new attendees on Days 2 and 3, respectively. The connections feature offered on the platform was the most utilized function within the online forum. Attendance in the parallel workshop sessions remained constant across the 3 days, with an average of 44.6% (SD = 6.77) of people moving between workshops within a single session. The two poster sessions had an average of 47.6 (SD = 17.97) and 27.8 (SD = 10.24) unique views per poster, respectively. Eleven percent ( N = 48) of attendees completed the post-conference survey. Thirty-six percent of these responders stated they were only able to attend because the conference was offered virtually. Further, the quality of the conference had an average satisfaction rating of 68.08 out of 100 (SD = 22.94)., Conclusion: Results of data analyses suggest the virtual platform allowed for those who were unable to attend to join virtually, produced moderate engagement throughout the conference, and that the majority of attendees were satisfied with the quality of the fully-virtual conference. Therefore, incorporating virtual aspects in future in-person conferences could enhance conference experience and participation., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Increased protein synthesis is a requirement for malignant growth, and as a result, translation has become a pharmaceutical target for cancer. The initiation of cap-dependent translation is enzymatically driven by the eukaryotic initiation factor (eIF)4A, an ATP-powered DEAD-box RNA-helicase that unwinds the messenger RNA secondary structure upstream of the start codon, enabling translation of downstream genes. A screen for inhibitors of eIF4A ATPase activity produced an intriguing hit that, surprisingly, was not ATP-competitive. A medicinal chemistry campaign produced the novel eIF4A inhibitor 28 , which decreased BJAB Burkitt lymphoma cell viability. Biochemical and cellular studies, molecular docking, and functional assays uncovered that 28 is an RNA-competitive, ATP-uncompetitive inhibitor that engages a novel pocket in the RNA groove of eIF4A and inhibits unwinding activity by interfering with proper RNA binding and suppressing ATP hydrolysis. Inhibition of eIF4A through this unique mechanism may offer new strategies for targeting this promising intersection point of many oncogenic pathways.

Native mass spectrometry (MS) has become a versatile tool for characterizing high-mass complexes and measuring biomolecular interactions. Native MS usually requires the resolution of different charge states produced by electrospray ionization to measure the mass, which is difficult for highly heterogeneous samples that have overlapping and unresolvable charge states. Charge detection-mass spectrometry (CD-MS) seeks to address this challenge by simultaneously measuring the charge and m / z for isolated ions. However, CD-MS often shows uncertainty in the charge measurement that limits the resolution. To overcome this charge state uncertainty, we developed UniDecCD (UCD) software for computational deconvolution of CD-MS data, which significantly improves the resolution of CD-MS data. Here, we describe the UCD algorithm and demonstrate its ability to improve the CD-MS resolution of proteins, megadalton viral capsids, and heterogeneous nanodiscs made from natural lipid extracts. UCD provides a user-friendly interface that will increase the accessibility of CD-MS technology and provide a valuable new computational tool for CD-MS data analysis.

NRF2 is a basic leucine zipper (bZip) transcription factor that is the master regulator of redox homeostasis. Under basal conditions, the cellular level of NRF2 is low due to a posttranslational regulation by the ubiquitin proteasome system (UPS). But, when an organism is challenged with oxidative or xenobiotic stress, the NRF2 pathway is activated by inhibition of the E3 ubiquitin ligase complex that normally marks NRF2 for destruction. For several decades, researchers have searched for molecules that can intentionally activate NRF2, as this was shown to be a means to prevent certain diseases, at least in animal models. In the present era, there are many compounds known to activate the NRF2 pathway including natural products and synthetic compounds, covalent and non-covalent compounds, and others. However, it was also revealed that like many protective pathways, the NRF2 pathway has a dark side. Just as NRF2 can protect normal cells from damage, it can protect malignant cells from damage. As cells transform, they are exposed to many stressors and aberrant upregulation of NRF2 can facilitate transformation and it can help cancer cells to grow, to spread, and to resist treatment. For this reason, researchers are also interested in the discovery and development of NRF2 inhibitors. In the present review, we will begin with a general discussion of NRF2 structure and function, we will discuss the latest in NRF2 non-covalent activators, and we will discuss the current state of NRF2 inhibitors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC 50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli , we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli , but only in the strict absence of either E. coli GroEL ( P. aeruginosa ) or both E. coli GroES and GroEL ( E. faecium ). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coli groESL was replaced with ESKAPE groESL , demonstrated similar growth kinetics to wild-type E. coli , but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity. IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coli groESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics., (Copyright © 2021 Sivinski et al.)

Objective: This study exploits the intersection between molecular-targeted therapies and immune-checkpoint inhibition to define new means to treat pancreatic cancer., Design: Patient-derived cell lines and xenograft models were used to define the response to CDK4/6 and MEK inhibition in the tumour compartment. Impacts relative to immunotherapy were performed using subcutaneous and orthotopic syngeneic models. Single-cell RNA sequencing and multispectral imaging were employed to delineate effects on the immunological milieu in the tumour microenvironment., Results: We found that combination treatment with MEK and CDK4/6 inhibitors was effective across a broad range of PDX models in delaying tumour progression. These effects were associated with stable cell-cycle arrest, as well as the induction of multiple genes associated with interferon response and antigen presentation in an RB-dependent fashion. Using single-cell sequencing and complementary approaches, we found that the combination of CDK4/6 and MEK inhibition had a significant impact on increasing T-cell infiltration and altering myeloid populations, while potently cooperating with immune checkpoint inhibitors., Conclusions: Together, these data indicate that there are canonical and non-canonical features of CDK4/6 and MEK inhibition that impact on the tumour and immune microenvironment. This combination-targeted treatment can promote robust tumour control in combination with immune checkpoint inhibitor therapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The identification of modulators for proteins without assayable biochemical activity remains a challenge in chemical biology. The presented approach adapts a high-throughput fluorescence binding assay and functional chromatography, two protein-resin technologies, enabling the discovery and isolation of fluorescent natural product probes that target proteins independently of biochemical function. The resulting probes also suggest targetable pockets for lead discovery. Using human survivin as a model, we demonstrate this method with the discovery of members of the prodiginine family as fluorescent probes to the cancer target survivin., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)