A Benzarone Derivative Inhibits EYA to Suppress Tumor Growth in SHH Medulloblastoma.

Medulloblastoma is one of the most common malignant brain tumors of children, and 30% of medulloblastomas are driven by gain-of-function genetic lesions in the Sonic Hedgehog (SHH) signaling pathway. EYA1, a haloacid dehalogenase phosphatase and transcription factor, is critical for tumorigenesis and proliferation of SHH medulloblastoma (SHH-MB). Benzarone and benzbromarone have been identified as allosteric inhibitors of EYA proteins. Using benzarone as a point of departure, we developed a panel of 35 derivatives and tested them in SHH-MB. Among these compounds, DS-1-38 functioned as an EYA antagonist and opposed SHH signaling. DS-1-38 inhibited SHH-MB growth in vitro and in vivo, showed excellent brain penetrance, and increased the lifespan of genetically engineered mice predisposed to fatal SHH-MB. These data suggest that EYA inhibitors represent promising therapies for pediatric SHH-MB. SIGNIFICANCE: Development of a benzarone derivative that inhibits EYA1 and impedes the growth of SHH medulloblastoma provides an avenue for improving treatment of this malignant pediatric brain cancer.

Tobacco-enhanced biofilm formation by Porphyromonas gingivalis and other oral microbes.

Microbial biofilms promote pathogenesis by disguising antigens, facilitating immune evasion, providing protection against antibiotics and other antimicrobials and, generally, fostering survival and persistence. Environmental fluxes are known to influence biofilm formation and composition, with recent data suggesting that tobacco and tobacco-derived stimuli are particularly important mediators of biofilm initiation and development in vitro and determinants of polymicrobial communities in vivo. The evidence for tobacco-augmented biofilm formation by oral bacteria, tobacco-induced oral dysbiosis, tobacco-resistance strategies, and bacterial physiology is summarized herein. A general overview is provided alongside specific insights gained through studies of the model and archetypal, anaerobic, Gram-negative oral pathobiont, Porphyromonas gingivalis.

Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma.

In pancreatic ductal adenocarcinoma (PDAC), glutamine is a critical nutrient that drives a wide array of metabolic and biosynthetic processes that support tumor growth. Here, we elucidate how 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that broadly inhibits glutamine metabolism, blocks PDAC tumor growth and metastasis. We find that DON significantly reduces asparagine production by inhibiting asparagine synthetase (ASNS), and that the effects of DON are rescued by asparagine. As a metabolic adaptation, PDAC cells upregulate ASNS expression in response to DON, and we show that ASNS levels are inversely correlated with DON efficacy. We also show that L-asparaginase (ASNase) synergizes with DON to affect the viability of PDAC cells, and that DON and ASNase combination therapy has a significant impact on metastasis. These results shed light on the mechanisms that drive the effects of glutamine mimicry and point to the utility of cotargeting adaptive responses to control PDAC progression.

A small molecule exerts selective antiviral activity by targeting the human cytomegalovirus nuclear egress complex.

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.

Asparagine restriction enhances CD8+ T cell metabolic fitness and antitumoral functionality through an NRF2-dependent stress response.

Robust and effective T cell immune surveillance and cancer immunotherapy require proper allocation of metabolic resources to sustain energetically costly processes, including growth and cytokine production. Here, we show that asparagine (Asn) restriction on CD8+ T cells exerted opposing effects during activation (early phase) and differentiation (late phase) following T cell activation. Asn restriction suppressed activation and cell cycle entry in the early phase while rapidly engaging the nuclear factor erythroid 2-related factor 2 (NRF2)-dependent stress response, conferring robust proliferation and effector function on CD8+ T cells during differentiation. Mechanistically, NRF2 activation in CD8+ T cells conferred by Asn restriction rewired the metabolic program by reducing the overall glucose and glutamine consumption but increasing intracellular nucleotides to promote proliferation. Accordingly, Asn restriction or NRF2 activation potentiated the T cell-mediated antitumoral response in preclinical animal models, suggesting that Asn restriction is a promising and clinically relevant strategy to enhance cancer immunotherapy. Our study revealed Asn as a critical metabolic node in directing the stress signaling to shape T cell metabolic fitness and effector functions.

A Real-Time Neurophysiologic Stress Test for the Aging Brain: Novel Perioperative and ICU Applications of EEG in Older Surgical Patients.

As of 2022, individuals age 65 and older represent approximately 10% of the global population [1], and older adults make up more than one third of anesthesia and surgical cases in developed countries [2, 3]. With approximately > 234 million major surgical procedures performed annually worldwide [4], this suggests that > 70 million surgeries are performed on older adults across the globe each year. The most common postoperative complications seen in these older surgical patients are perioperative neurocognitive disorders including postoperative delirium, which are associated with an increased risk for mortality [5], greater economic burden [6, 7], and greater risk for developing long-term cognitive decline [8] such as Alzheimer's disease and/or related dementias (ADRD). Thus, anesthesia, surgery, and postoperative hospitalization have been viewed as a biological "stress test" for the aging brain, in which postoperative delirium indicates a failed stress test and consequent risk for later cognitive decline (see Fig. 3). Further, it has been hypothesized that interventions that prevent postoperative delirium might reduce the risk of long-term cognitive decline. Recent advances suggest that rather than waiting for the development of postoperative delirium to indicate whether a patient "passed" or "failed" this stress test, the status of the brain can be monitored in real-time via electroencephalography (EEG) in the perioperative period. Beyond the traditional intraoperative use of EEG monitoring for anesthetic titration, perioperative EEG may be a viable tool for identifying waveforms indicative of reduced brain integrity and potential risk for postoperative delirium and long-term cognitive decline. In principle, research incorporating routine perioperative EEG monitoring may provide insight into neuronal patterns of dysfunction associated with risk of postoperative delirium, long-term cognitive decline, or even specific types of aging-related neurodegenerative disease pathology. This research would accelerate our understanding of which waveforms or neuronal patterns necessitate diagnostic workup and intervention in the perioperative period, which could potentially reduce postoperative delirium and/or dementia risk. Thus, here we present recommendations for the use of perioperative EEG as a "predictor" of delirium and perioperative cognitive decline in older surgical patients.

Perioperative anaphylaxis management and testing: a quaternary centre audit.

BACKGROUND: Anaphylaxis is a severe, potentially life-threatening generalized or systemic hypersensitivity reaction. Sequential reports have cited anaphylaxis as the most common cause of anaesthesia-related deaths. We undertook an audit at a quaternary centre, examining the management of perioperative anaphylaxis and quality of referrals made to our anaesthesia allergy testing service. METHODS: The data of 41 patients consulted at St Vincent's Hospital Melbourne for perioperative anaphylaxis between 17th of January 2020 and 20th of January 2022 were analysed. Intervention outcomes included total intravenous fluid administered, adrenaline administration, instigation of CPR and the collection and the timing of serum tryptase samples. We also assessed referral quality, provision of institutional allergy alert and time elapsed from the anaphylaxis event to allergy testing. Contemporaneous Australian and New Zealand Anaesthetic Allergy Group (ANZAAG) guidelines were used as the reference standard for most outcomes. RESULTS: Our data reveals compliance of <80% with respect to intravenous fluid administration, referral quality and tryptase sampling, particularly at the 4-h timepoint. CONCLUSION: Surgical leadership and patient advocacy in the post-acute phase would likely facilitate requisite testing and improve the quality of counselling. We recommend institutions adopt a case-by-case review of management compliance with recommendations. Additionally, we advocate for the inclusion of a prompt to the ANZAAG referral form, that encourages the operator to update their patient's institutional allergy alert while awaiting allergy testing.

Examining Subjective Psychological Experiences of Postoperative Delirium in Older Cardiac Surgery Patients.

BACKGROUND: Postoperative delirium (POD) is an acute syndrome including inattention and impaired cognition that affects approximately 42% of older cardiac surgical patients. POD is linked to adverse outcomes including morbidity, mortality, and further cognitive decline. Less is known about the subjective psychological experience of POD and its ongoing impact on well-being. METHODS: We performed a qualitative analysis of the long-term psychological sequelae of older adults who experience POD after cardiac surgery. We sampled 30 patients aged 60 years and older who experienced at least 2 episodes of POD during a prior hospital admission. We administered semistructured interviews with participants via telephone 3 to 5 years postoperatively. Interviews were transcribed and thematically analyzed. Data were interpreted in accordance with the naturalist paradigm. RESULTS: Three overarching themes emerged in our qualitative analysis. The first reflected the multifaceted presentation of POD, including distortion of time and reality; feelings of isolation; and a loss of self, identity, and control. The second theme reflected the psychological challenges associated with functional decline after surgery. Common examples of functional decline included cognitive difficulties, excessive fatigue, and a perceived loss of independence. The final theme captured the emotional sequelae of acute illness, which included low mood, reduced motivation, and social comparisons. CONCLUSIONS: Our findings emphasize the multidimensional experience of POD and long-term effects on psychological wellbeing. Our research highlights the beneficial role multidisciplinary clinicians play in managing POD including strategies that may be embedded into clinical practice and helps anesthesiologists understand why patients who have experienced POD in the past may present with specific concerns should they require subsequent surgery.

Tobacco smoke exacerbates Filifactor alocis pathogenicity.

AIM: Filifactor alocis has recently emerged as a periodontal pathobiont that appears to thrive in the oral cavity of smokers. We hypothesized that identification of smoke-responsive F. alocis genes would provide insight into adaptive strategies and that cigarette smoke would enhance F. alocis pathogenesis in vivo. MATERIALS AND METHODS: F. alocis was grown in vitro and cigarette smoke extract-responsive genes determined by RNAseq. Mice were exposed, or not, to mainstream 1R6F research cigarette smoke and infected with F. alocis, or not, in an acute ligature model of periodontitis. Key clinical, infectious, and immune data were collected. RESULTS: In culture, F. alocis growth was unaffected by smoke conditioning and only a small number of genes were specifically regulated by smoke exposure. Reduced murine mass, differences in F. alocis-cognizant antibody production, and altered immune profiles as well as altered alveolar bone loss were all attributable to smoke exposure and/or F. alocis infection in vivo. CONCLUSIONS: F. alocis is well-adapted to tobacco-rich conditions and its pathogenesis is enhanced by tobacco smoke exposure. A smoke-exposed ligature model of periodontitis shows promise as a tool with which to further unravel mechanisms underlying tobacco-enhanced, bacteria-induced disease.

Health-related quality of life after restrictive versus liberal RBC transfusion for cardiac surgery: Sub-study from a randomized clinical trial.

BACKGROUND: Transfusion Requirements in Cardiac Surgery III (TRICS III), a multi-center randomized controlled trial, demonstrated clinical non-inferiority for restrictive versus liberal RBC transfusion for patients undergoing cardiac surgery. However, it is uncertain if transfusion strategy affects long-term health-related quality of life (HRQOL). STUDY DESIGN AND METHODS: In this planned sub-study of Australian patients in TRICS III, we sought to determine the non-inferiority of restrictive versus liberal transfusion strategy on long-term HRQOL and to describe clinical outcomes 24 months postoperatively. The restrictive strategy involved transfusing RBCs when hemoglobin was <7.5 g/dl; the transfusion triggers in the liberal group were: <9.5 g/L intraoperatively, <9.5 g/L in intensive care, or <8.5 g/dl on the ward. HRQOL assessments were performed using the 36-item short form survey version 2 (SF-36v2). Primary outcome was non-inferiority of summary measures of SF-36v2 at 12 months, (non-inferiority margin: -0.25 effect size; restrictive minus liberal scores). Secondary outcomes included non-inferiority of HRQOL at 18 and 24 months. RESULTS: Six hundred seventeen Australian patients received allocated randomization; HRQOL data were available for 208/311 in restrictive and 217/306 in liberal group. After multiple imputation, non-inferiority of restrictive transfusion at 12 months was not demonstrated for HRQOL, and the estimates were directionally in favor of liberal transfusion. Non-inferiority also could not be concluded at 18 and 24 months. Sensitivity analyses supported these results. There were no differences in quality-adjusted life years or composite clinical outcomes up to 24 months after surgery. DISCUSSION: The non-inferiority of a restrictive compared to a liberal transfusion strategy was not established for long-term HRQOL in this dataset.

Atypical cyclic di-AMP signaling is essential for Porphyromonas gingivalis growth and regulation of cell envelope homeostasis and virulence.

Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3',5')-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pdepg) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pdepg and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe-microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.

A stromal Integrated Stress Response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumour progression.

Bidirectional signalling between the tumour and stroma shapes tumour aggressiveness and metastasis. ATF4 is a major effector of the Integrated Stress Response, a homeostatic mechanism that couples cell growth and survival to bioenergetic demands. Using conditional knockout ATF4 mice, we show that global, or fibroblast-specific loss of host ATF4, results in deficient vascularization and a pronounced growth delay of syngeneic melanoma and pancreatic tumours. Single-cell transcriptomics of tumours grown in Atf4Δ/Δ mice uncovered a reduction in activation markers in perivascular cancer-associated fibroblasts (CAFs). Atf4Δ/Δ fibroblasts displayed significant defects in collagen biosynthesis and deposition and a reduced ability to support angiogenesis. Mechanistically, ATF4 regulates the expression of the Col1a1 gene and levels of glycine and proline, the major amino acids of collagen. Analyses of human melanoma and pancreatic tumours revealed a strong correlation between ATF4 and collagen levels. Our findings establish stromal ATF4 as a key driver of CAF functionality, malignant progression and metastasis.

Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors.

Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy. We identify a subset of irreversible EGFR inhibitors that display positive binding cooperativity and synergy with the allosteric inhibitor JBJ-04-125-02 in several EGFR variants. Structural analysis of these complexes reveals conformational changes occur mainly in the phosphate-binding loop (P-loop). Mutation of F723 in the P-loop reduces cooperative binding and synergy, supporting a mechanism in which F723-mediated contacts between the P-loop and the allosteric inhibitor are critical for synergy. These structural and mechanistic insights will aid in the identification and development of additional inhibitor combinations with potential clinical value.

Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.

Quinazolinones as allosteric fourth-generation EGFR inhibitors for the treatment of NSCLC.

The C797S mutation confers resistance to covalent EGFR inhibitors used in the treatment of lung tumors with the activating L858R mutation. Isoindolinones such as JBJ-4-125-02 bind in an allosteric pocket and are active against this mutation, with high selectivity over wild-type EGFR. The most potent examples we developed from that series have a potential chemical instability risk from the combination of the amide and phenol groups. We explored a scaffold hopping approach to identify new series of allosteric EGFR inhibitors that retained good potency in the absence of the phenol group. The 5-F quinazolinone 34 demonstrated tumor regression in an H1975 efficacy model upon once daily oral dosing at 25 mg/kg.

An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.

Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By using complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fueled biosynthetic processes while controlling the ratio of succinate/α-ketoglutarate (α-KG) to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells caused proliferation and survival defects when the TCA cycle was truncated, blocking carbon flux to support nucleoside biosynthesis. Replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. SDH deficiency induced a proinflammatory gene signature in T cells and promoted T helper 1 and T helper 17 lineage differentiation. An increasing succinate/α-KG ratio in SDH-deficient T cells promoted inflammation by changing the pattern of the transcriptional and chromatin accessibility signatures and consequentially increasing the expression of the transcription factor, PR domain zinc finger protein 1. Collectively, our studies revealed a role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

Comparing the effect of xenon and sevoflurane anesthesia on postoperative neural injury biomarkers: a randomized controlled trial.

General anesthesia and surgery are associated with an increase in neural injury biomarkers. Elevations of these neural injury biomarkers in the perioperative period are associated with postoperative delirium. Xenon has been shown to be protective against a range of neurological insults in animal models. It remains to be seen if xenon anesthesia is neuroprotective in the perioperative setting in humans. Twenty-four participants scheduled for lithotripsy were randomized to receive either xenon or sevoflurane general anesthesia. There was no statistically significant difference in the concentrations of postoperative neural injury biomarkers between the xenon and sevoflurane group. Following the procedure there was a significant increase in the concentration from baseline of all three biomarkers at 1 hour post-induction with a return to baseline at 5 hours. General anesthesia for lithotripsy was associated with a significant increase at 1 hour post-induction in the neural injury biomarkers total tau, neurofilament light and tau phosphorylated at threonine 181, a marker of tau phosphorylation. The protocol was approved by the St. Vincent's Hospital Melbourne Ethics Committee (approval No. HREC/18/SVHM/221) on July 20, 2018 and was registered with the Australia New Zealand Clinical Trials Registry (registration No. ACTRN12618000916246) on May 31, 2018.