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The proteasome, the catalytic arm of the ubiquitin system, is regulated via its dynamic compartmentation between the nucleus and the cytoplasm, among other mechanisms. Under amino acid shortage, the proteolytic complex is translocated to the cytoplasm, where it stimulates proteolysis to supplement recycled amino acids for essential protein synthesis. This response is mediated via the mTOR pathway and the lack of the three aromatic amino acids Tyr, Trp, and Phe (YWF). mTOR activation by supplementation of the triad inhibits proteasome translocation, leading to cell death. We now show that tumoral inherent stress conditions result in translocation of the proteasome from the nucleus to the cytosol. We further show that the modulation of the signaling cascade governed by YWF is applicable also to non-starved cells by using higher concentration of the triad to achieve a surplus relative to all other amino acids. Based on these two phenomena, we found that the modulation of stress signals via the administration of YWF leads to nuclear proteasome sequestration and inhibition of growth of xenograft, spontaneous, and metastatic mouse tumor models. In correlation with the observed effect of YWF on tumors, we found - using transcriptomic and proteomic analyses - that the triad affects various cellular processes related to cell proliferation, migration, and death. In addition, Sestrin3-a mediator of YWF sensing upstream of mTOR-is essential for proteasome translocation, and therefore plays a pro-tumorigenic role, positioning it as a potential oncogene. This newly identified approach for hijacking the cellular "satiety center" carries therefore potential therapeutic implications for cancer., (© 2024. The Author(s).)

Objectives: To ascertain the success of endo-DCR in nasolacrimal duct stenosis (NLDS) versus nasolacrimal duct obstruction (NLDO)., Methods: Consecutive adult patients with epiphora attending a tertiary lacrimal clinic from February 2012 to February 2021 were reviewed. NLDS was diagnosed by patent lacrimal syringing and combined dacryocystography (NLD stenosis) and dacryoscintigraphy (post-sac delay) findings in all eyes. Cases with evidence of canalicular stenosis or other identifiable causes of epiphora were excluded. The epiphora resolution and improvement rates following endo-DCR were compared between NLDS and complete NLDO cases., Results: DCRs in 24 NLDS (23 patients, 69.6% females, mean age 61.0 ± 17.07) and 58 NLDO (56 patients, 69.6% females, mean age 61.9 ± 17.4) were included. Resolution of epiphora was achieved in 10 (41.7% [95% CI 0.24-0.61]) of the NLDS cases compared to 40 (69.0% [95% CI 0.56-0.79]) in NLDO (p = 0.021). Improvement of epiphora (i.e., either improvement or resolution) was noted in 17 (70.8% [95% CI 0.51-0.85]) of NLDS and 53 (91.4% [95% CI 0.81-0.96]) of NLDO cases (p = 0.034). Three patients (12.5%) with NLDS had subsequent lacrimal procedures (one DCR revision, two Jones tube) at a median of 14 (range 11-21) months. 71.4% of the NLDS patients responded to a phone questionnaire at a median of 93 months postoperatively. Of these, 46.7% reported resolution or significant improvement, and 33.3% reported slight improvement. 64.3% said they would recommend DCR to others suffering from epiphora., Conclusion: Endo-DCR may benefit approximately 70% of patients with NLDS. The success of endo-DCR in complete NLDO may be higher., (© 2022. The Author(s).)

Objectives: To describe the features, management approaches, and outcomes of orbito-cranial schwannomas., Methods: Retrospective review of ten patients with orbito-cranial schwannomas managed in six orbital services over 22 years. Data collected included demographics, presenting features, neuroimaging characteristics, histology, management approach, complications, and outcomes., Results: Mean age of the patients was 41.4 ± 19.9 years, and 6 (60%) were females. The majority presented with proptosis (90%), limited extraocular motility (80%), eyelid swelling (60%), and optic neuropathy (60%). Most lesions (80%) involved the entire anterior-posterior span of the orbit, with both intra- and extraconal involvement. All tumours involved the orbital apex, the superior orbital fissure, and extended at least to the cavernous sinus. Surgical resection was performed for all. Seven (70%) of the tumours were completely or subtotally resected combining an intracapsular approach by an orbital-neurosurgical collaboration, with no recurrence on postoperative follow-up (6-186 months). Three underwent tumour debulking. Of these, two remained stable on follow-up (6-34 months) and one showed progression of the residual tumour over 9 years (cellular schwannoma on histology) necessitating stereotactic radiotherapy (SRT) for local control. Adjuncts to the orbito-cranial resection included perioperative frozen section (n = 5), endoscopic transorbital approach (n = 2), and image-guided navigation (n = 1). Post-surgical adjuvant SRT was used in three subjects., Conclusions: These results highlight the possibility of successful surgical control in complex orbito-cranial schwannomas. A combined neurosurgical/orbital approach with consideration of an intracapsular resection is recommended. Recurrence may not occur with subtotal excision and observation may be reasonable. Adjunctive SRT for progression or residual tumour can be considered., (© 2021. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Objectives: To report associations with visual function and quality of life (QOL) in artificial eye wearers., Methods: Multicentre, observational, cross-sectional, nationwide study, within the National Health Service England. Items were adopted from the National Eye Institute Visual Function Questionnaire, and incorporated in the National Artificial Eye Questionnaire (NAEQ). The NAEQ was completed by 951 respondents. Multiple regressions assessed associations between the QOL scores and the experiences of artificial eye wearers, their routine management, changes over time, baseline and demographic parameters., Results: Parameters predictive of a better QOL composite score included longer artificial eye wear (β = 0.18, p < 0.001), better appearance (β = 0.17, p < 0.001), better comfort (β = 0.14, p = 0.001), tumour-related anophthalmia (β = 0.13, p = 0.003), male gender (β = 0.13, p < 0.001), shorter period of adjustment to monocular vision (β = 0.12, p < 0.001) and use of soap for cleaning (β = 0.09, p = 0.046). The composite score continued to improve beyond 10 years of prosthesis wear (≤2 years mean 72.80 ± 1.65 versus >10 years mean 79.45 ± 0.70; p = 0.001). Both better prosthesis appearance (β = 0.14, p = 0.022) and improved motility (β = 0.13, p = 0.042) predicted a better dependency score. Use of lubricating ointment predicted a worse dependency score (β = 0.23, p = 0.003). Neither the frequency of removal, nor the cleaning frequency of the artificial eye correlated with QOL scales., Conclusions: Multiple factors in the artificial eye experience were found to predict visual function and QOL aspects. This study underscores the need to generate a dedicated QOL questionnaire for use in anophthalmic patients., (© 2021. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

SARS-CoV-2 variants of interest and concern will continue to emerge for the duration of the COVID-19 pandemic. To map mutations in the receptor-binding domain (RBD) of the spike protein that affect binding to angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, we applied in vitro evolution to affinity-mature the RBD. Multiple rounds of random mutagenic libraries of the RBD were sorted against decreasing concentrations of ACE2, resulting in the selection of higher affinity RBD binders. We found that mutations present in more transmissible viruses (S477N, E484K and N501Y) were preferentially selected in our high-throughput screen. Evolved RBD mutants include prominently the amino acid substitutions found in the RBDs of B.1.620, B.1.1.7 (Alpha), B1.351 (Beta) and P.1 (Gamma) variants. Moreover, the incidence of RBD mutations in the population as presented in the GISAID database (April 2021) is positively correlated with increased binding affinity to ACE2. Further in vitro evolution increased binding by 1,000-fold and identified mutations that may be more infectious if they evolve in the circulating viral population, for example, Q498R is epistatic to N501Y. We show that our high-affinity variant RBD-62 can be used as a drug to inhibit infection with SARS-CoV-2 and variants Alpha, Beta and Gamma in vitro. In a model of SARS-CoV-2 challenge in hamster, RBD-62 significantly reduced clinical disease when administered before or after infection. A 2.9 Å cryo-electron microscopy structure of the high-affinity complex of RBD-62 and ACE2, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: To report associations with comfort and with appearance satisfaction in artificial eye wearers., Methods: Multicentre, observational, cross-sectional study, nationwide within the National Health Service England. The National Artificial Eye Questionnaire (NAEQ) was completed by 951 respondents. Multiple regressions assessed associations between the experiences of artificial eye wearers, routine management, changes over time, baseline and demographic parameters and their reported comfort, satisfaction with appearance and prosthesis motility., Results: Better comfort levels were associated with needing less lubrication (β = 0.24, p < 0.001), older age (β = 0.17, p = 0.014), less discharge (β = 0.16, p < 0.001), less frequent cleaning (β = 0.16, p = 0.043), and male gender (β = 0.06, p = 0.047). Greater satisfaction with the appearance of the artificial eye was associated with better perceived motility (β = 0.57, p < 0.001). Black ethnic origin predicted a lower satisfaction with the appearance (β = -0.17, p = 0.001). Greater satisfaction with the motility was associated with a better appearance rating (β = 0.51, p < 0.001), longer time of having an artificial eye (β = 0.13, p < 0.001), older age (β = 0.11, p = 0.042), and a shorter adjustment time (β = -0.07, p = 0.016). Of the testimonials concerning appearance aspects, the majority (21/45, 46.7%) were related to the effect on social interactions., Conclusions: The results suggest that more attention should be given to the "dry anophthalmic socket syndrome" as a key cause of discomfort. Young patients are concerned particularly about the motility of the artificial eye. Over time satisfaction with the artificial eye movement is likely to improve., (© 2020. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Globally consistent measurements of airborne metal concentrations in fine particulate matter (PM 2.5 ) are important for understanding potential health impacts, prioritizing air pollution mitigation strategies, and enabling global chemical transport model development. PM 2.5 filter samples (N ~ 800 from 19 locations) collected from a globally distributed surface particulate matter sampling network (SPARTAN) between January 2013 and April 2019 were analyzed for particulate mass and trace metals content. Metal concentrations exhibited pronounced spatial variation, primarily driven by anthropogenic activities. PM 2.5 levels of lead, arsenic, chromium, and zinc were significantly enriched at some locations by factors of 100-3000 compared to crustal concentrations. Levels of metals in PM 2.5 and PM 10 exceeded health guidelines at multiple sites. For example, Dhaka and Kanpur sites exceeded the US National Ambient Air 3-month Quality Standard for lead (150 ng m -3 ). Kanpur, Hanoi, Beijing and Dhaka sites had annual mean arsenic concentrations that approached or exceeded the World Health Organization's risk level for arsenic (6.6 ng m -3 ). The high concentrations of several potentially harmful metals in densely populated cites worldwide motivates expanded measurements and analyses.

Various respiratory viral infections in general and seasonal influenza in particular may increase the susceptibility to bacterial infections. Plague caused by Yersinia pestis endangers large populations during outbreaks or bioterrorism attacks. Recommended antibiotic countermeasures include well-established protocols based on animal studies and corroborated by effective treatment of human cases. Until now, prior exposure to viral respiratory infections was not taken into consideration when selecting the appropriate treatment for plague. Here, we show that as late as 25 days after exposure to influenza virus, convalescent mice still exhibited an increased susceptibility to sublethal doses of Y. pestis, presented with aberrant cytokine expression, and impaired neutrophil infiltration in the lungs. Increased levels of M2 alveolar macrophages and type II epithelial cells, as well as induction in metalloproteases expression and collagen and laminin degradation, suggested that the previous viral infection was under resolution, correlating with enhanced susceptibility to plague. Surprisingly, postexposure prophylaxis treatment with the recommended drugs revealed that ciprofloxacin was superior to doxycycline in mice recovering from influenza infection. These results suggest that after an influenza infection, the consequences, such as impaired immunity and lung tissue remodeling and damage, should be considered when treating subsequent Y. pestis exposure.

Somatic mutations in p53, which inactivate the tumour-suppressor function of p53 and often confer oncogenic gain-of-function properties, are very common in cancer 1,2 . Here we studied the effects of hotspot gain-of-function mutations in Trp53 (the gene that encodes p53 in mice) in mouse models of WNT-driven intestinal cancer caused by Csnk1a1 deletion 3,4 or Apc Min mutation 5 . Cancer in these models is known to be facilitated by loss of p53 3,6 . We found that mutant versions of p53 had contrasting effects in different segments of the gut: in the distal gut, mutant p53 had the expected oncogenic effect; however, in the proximal gut and in tumour organoids it had a pronounced tumour-suppressive effect. In the tumour-suppressive mode, mutant p53 eliminated dysplasia and tumorigenesis in Csnk1a1-deficient and Apc Min/+ mice, and promoted normal growth and differentiation of tumour organoids derived from these mice. In these settings, mutant p53 was more effective than wild-type p53 at inhibiting tumour formation. Mechanistically, the tumour-suppressive effects of mutant p53 were driven by disruption of the WNT pathway, through preventing the binding of TCF4 to chromatin. Notably, this tumour-suppressive effect was completely abolished by the gut microbiome. Moreover, a single metabolite derived from the gut microbiota-gallic acid-could reproduce the entire effect of the microbiome. Supplementing gut-sterilized p53-mutant mice and p53-mutant organoids with gallic acid reinstated the TCF4-chromatin interaction and the hyperactivation of WNT, thus conferring a malignant phenotype to the organoids and throughout the gut. Our study demonstrates the substantial plasticity of a cancer mutation and highlights the role of the microenvironment in determining its functional outcome.

P-glycoprotein (P-gp), an ATP-dependent efflux pump, is associated with the development of multidrug resistance in cancer cells. Antibody-mediated blockade of human P-gp activity has been shown to overcome drug resistance by re-sensitizing resistant cancer cells to anticancer drugs. Despite the potential clinical application of this finding, the epitopes of the three human P-gp-specific monoclonal antibodies MRK-16, UIC2 and 4E3, which bind to the extracellular loops (ECLs) have not yet been mapped. By generating human-mouse P-gp chimeras, we mapped the epitopes of these antibodies to ECLs 1 and 4. We then identified key amino acids in these regions by replacing mouse residues with homologous human P-gp residues to recover binding of antibodies to the mouse P-gp. We found that changing a total of ten residues, five each in ECL1 and ECL4, was sufficient to recover binding of both MRK-16 and 4E3 antibodies, suggesting a common epitope. However, recovery of the conformation-sensitive UIC2 epitope required replacement of thirteen residues in ECL1 and the same five residues replaced in the ECL4 for MRK-16 and 4E3 binding. These results demonstrate that discontinuous epitopes for MRK-16, UIC2 and 4E3 are located in the same regions of ECL1 and 4 of the multidrug transporter.

The intestinal epithelium holds an immense regenerative capacity mobilized by intestinal stem cells (ISCs), much of it supported by Wnt pathway activation. Several unique regulatory mechanisms ensuring optimal levels of Wnt signaling have been recognized in ISCs. Here, we identify another Wnt signaling amplifier, CKIε, which is specifically upregulated in ISCs and is essential for ISC maintenance, especially in the absence of its close isoform CKIδ. Co-ablation of CKIδ/ε in the mouse gut epithelium results in rapid ISC elimination, with subsequent growth arrest, crypt-villous shrinking, and rapid mouse death. Unexpectedly, Wnt activation is preserved in all CKIδ/ε-deficient enterocyte populations, with the exception of Lgr5 + ISCs, which exhibit Dvl2-dependent Wnt signaling attenuation. CKIδ/ε-depleted gut organoids cease proliferating and die rapidly, yet survive and resume self-renewal upon reconstitution of Dvl2 expression. Our study underscores a unique regulation mode of the Wnt pathway in ISCs, possibly providing new means of stem cell enrichment for regenerative medicine., (© 2017 The Authors.)

Secondary organic aerosol (SOA) accounts for a dominant fraction of the submicron atmospheric particle mass, but knowledge of the formation, composition and climate effects of SOA is incomplete and limits our understanding of overall aerosol effects in the atmosphere. Organic oligomers were discovered as dominant components in SOA over a decade ago in laboratory experiments and have since been proposed to play a dominant role in many aerosol processes. However, it remains unclear whether oligomers are relevant under ambient atmospheric conditions because they are often not clearly observed in field samples. Here we resolve this long-standing discrepancy by showing that elevated SOA mass is one of the key drivers of oligomer formation in the ambient atmosphere and laboratory experiments. We show for the first time that a specific organic compound class in aerosols, oligomers, is strongly correlated with cloud condensation nuclei (CCN) activities of SOA particles. These findings might have important implications for future climate scenarios where increased temperatures cause higher biogenic volatile organic compound (VOC) emissions, which in turn lead to higher SOA mass formation and significant changes in SOA composition. Such processes would need to be considered in climate models for a realistic representation of future aerosol-climate-biosphere feedbacks.

The mature gut renews continuously and rapidly throughout adult life, often in a damage-inflicting micro-environment. The major driving force for self-renewal of the intestinal epithelium is the Wnt-mediated signalling pathway, and Wnt signalling is frequently hyperactivated in colorectal cancer. Here we show that casein kinase Iα (CKIα), a component of the β-catenin-destruction complex, is a critical regulator of the Wnt signalling pathway. Inducing the ablation of Csnk1a1 (the gene encoding CKIα) in the gut triggers massive Wnt activation, surprisingly without causing tumorigenesis. CKIα-deficient epithelium shows many of the features of human colorectal tumours in addition to Wnt activation, in particular the induction of the DNA damage response and cellular senescence, both of which are thought to provide a barrier against malignant transformation. The epithelial DNA damage response in mice is accompanied by substantial activation of p53, suggesting that the p53 pathway may counteract the pro-tumorigenic effects of Wnt hyperactivation. Notably, the transition from benign adenomas to invasive colorectal cancer in humans is typically linked to p53 inactivation, underscoring the importance of p53 as a safeguard against malignant progression; however, the mechanism of p53-mediated tumour suppression is unknown. We show that the maintenance of intestinal homeostasis in CKIα-deficient gut requires p53-mediated growth control, because the combined ablation of Csnk1a1 and either p53 or its target gene p21 (also known as Waf1, Cip1, Sdi1 and Cdkn1a) triggered high-grade dysplasia with extensive proliferation. Unexpectedly, these ablations also induced non-proliferating cells to invade the villous lamina propria rapidly, producing invasive carcinomas throughout the small bowel. Furthermore, in p53-deficient gut, loss of heterozygosity of the gene encoding CKIα caused a highly invasive carcinoma, indicating that CKIα functions as a tumour suppressor when p53 is inactivated. We identified a set of genes (the p53-suppressed invasiveness signature, PSIS) that is activated by the loss of both p53 and CKIα and which probably accounts for the brisk induction of invasiveness. PSIS transcription and tumour invasion were suppressed by p21, independently of cell cycle control. Restraining tissue invasion through suppressing PSIS expression is thus a novel tumour-suppressor function of wild-type p53.

Two major arms of the inflammatory response are the NF-κB and c-Jun N-terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn-dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF-κB and AP-1 activation. We show that NleC and NleD co-operate and complement other EPEC effectors in accomplishing maximal inhibition of IL-8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.

The causes of sporadic human cancer are seldom recognized, but it is estimated that carcinogen exposure and chronic inflammation are two important underlying conditions for tumour development, the latter accounting for approximately 20% of human cancer. Whereas the causal relationship between carcinogen exposure and cancer has been intensely investigated, the molecular and cellular mechanisms linking chronic inflammation to tumorigenesis remain largely unresolved. We proposed that activation of the nuclear factor kappaB (NF-kappaB), a hallmark of inflammatory responses that is frequently detected in tumours, may constitute a missing link between inflammation and cancer. To test this hypothesis, we studied the Mdr2-knockout mouse strain, which spontaneously develops cholestatic hepatitis followed by hepatocellular carcinoma, a prototype of inflammation-associated cancer. We monitored hepatitis and cancer progression in Mdr2-knockout mice, and here we show that the inflammatory process triggers hepatocyte NF-kappaB through upregulation of tumour-necrosis factor-alpha (TNFalpha) in adjacent endothelial and inflammatory cells. Switching off NF-kappaB in mice from birth to seven months of age, using a hepatocyte-specific inducible IkappaB-super-repressor transgene, had no effect on the course of hepatitis, nor did it affect early phases of hepatocyte transformation. By contrast, suppressing NF-kappaB inhibition through anti-TNFalpha treatment or induction of IkappaB-super-repressor in later stages of tumour development resulted in apoptosis of transformed hepatocytes and failure to progress to hepatocellular carcinoma. Our studies thus indicate that NF-kappaB is essential for promoting inflammation-associated cancer, and is therefore a potential target for cancer prevention in chronic inflammatory diseases.