Incidence and predictors of thermal oesophageal and vagus nerve injuries in Ablation Index-guided high-power-short-duration ablation of atrial fibrillation: a prospective study.

AIMS: High-power-short-duration (HPSD) ablation is an effective treatment for atrial fibrillation but poses risks of thermal injuries to the oesophagus and vagus nerve. This study aims to investigate incidence and predictors of thermal injuries, employing machine learning. METHODS AND RESULTS: A prospective observational study was conducted at Leipzig Heart Centre, Germany, excluding patients with multiple prior ablations. All patients received Ablation Index-guided HPSD ablation and subsequent oesophagogastroduodenoscopy. A machine learning algorithm categorized ablation points by atrial location and analysed ablation data, including Ablation Index, focusing on the posterior wall. The study is registered in clinicaltrials.gov (NCT05709756). Between February 2021 and August 2023, 238 patients were enrolled, of whom 18 (7.6%; nine oesophagus, eight vagus nerve, one both) developed thermal injuries, including eight oesophageal erythemata, two ulcers, and no fistula. Higher mean force (15.8 ± 3.9 g vs. 13.6 ± 3.9 g, P = 0.022), ablation point quantity (61.50 ± 20.45 vs. 48.16 ± 19.60, P = 0.007), and total and maximum Ablation Index (24 114 ± 8765 vs. 18 894 ± 7863, P = 0.008; 499 ± 95 vs. 473 ± 44, P = 0.04, respectively) at the posterior wall, but not oesophagus location, correlated significantly with thermal injury occurrence. Patients with thermal injuries had significantly lower distances between left atrium and oesophagus (3.0 ± 1.5 mm vs. 4.4 ± 2.1 mm, P = 0.012) and smaller atrial surface areas (24.9 ± 6.5 cm2 vs. 29.5 ± 7.5 cm2, P = 0.032). CONCLUSION: The low thermal lesion's rate (7.6%) during Ablation Index-guided HPSD ablation for atrial fibrillation is noteworthy. Machine learning based ablation data analysis identified several potential predictors of thermal injuries. The correlation between machine learning output and injury development suggests the potential for a clinical tool to enhance procedural safety.

Repair-independent functions of DNA-PKcs protect irradiated cells from mitotic slippage and accelerated senescence.

The binding of DNA-dependent protein kinase catalytic subunit (DNA-PKcs, also known as PRKDC) to Ku proteins at DNA double-strand breaks (DSBs) has long been considered essential for non-homologous end joining (NHEJ) repair, providing a rationale for use of DNA-PKcs inhibitors as cancer therapeutics. Given lagging clinical translation, we reexamined mechanisms and observed instead that DSB repair can proceed independently of DNA-PKcs. While repair of radiation-induced DSBs was blocked in cells expressing shRNAs targeting Ku proteins or other NHEJ core factors, DSBs were repaired on schedule despite targeting DNA-PKcs. Although we failed to observe a DSB repair defect, the γH2AX foci that formed at sites of DNA damage persisted indefinitely after irradiation, leading to cytokinesis failure and accumulation of binucleated cells. Following this mitotic slippage, cells with decreased DNA-PKcs underwent accelerated cellular senescence. We identified downregulation of ataxia-telangiectasia mutated kinase (ATM) as the critical role of DNA-PKcs in recovery from DNA damage, insofar as targeting ATM restored γH2AX foci resolution and cytokinesis. Considering the lack of direct impact on DSB repair and emerging links between senescence and resistance to cancer therapy, these results suggest reassessing DNA-PKcs as a target for cancer treatment.

Alkane and wax ester production from lignin-related aromatic compounds.

Lignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in the industry. Here, we investigated the properties of lignin-related aromatic compounds (LRAs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in an LRA-utilizing bacterial host Acinetobacter baylyi ADP1. The biosynthesis products, wax esters, and alkanes are relevant compounds for the chemical and fuel industries. Here, wax esters were produced by a native pathway of ADP1, whereas alkanes were produced by a synthetic pathway introduced to the host. Using individual LRAs as substrates, the growth and product formation were monitored with internal biosensors and off-line analytics. Of the tested LRAs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with yield and titer of 37 mg/gcoumarate and 202 mg/L, whereas alkanes were produced with a yield of 62.3 µg /gcoumarate and titer of 152 µg/L. This study demonstrates the microbial preference for certain LRAs and highlights the potential of A. baylyi ADP1 as a host for LRA upgrading to value-added products.

Synthetic metabolic pathway for the production of 1-alkenes from lignin-derived molecules.

BACKGROUND: Integration of synthetic metabolic pathways to catabolically diverse chassis provides new opportunities for sustainable production. One attractive scenario is the use of abundant waste material to produce a readily collectable product, which can reduce the production costs. Towards that end, we established a cellular platform for the production of semivolatile medium-chain α-olefins from lignin-derived molecules: we constructed 1-undecene synthesis pathway in Acinetobacter baylyi ADP1 using ferulate, a lignin-derived model compound, as the sole carbon source for both cell growth and product synthesis. RESULTS: In order to overcome the toxicity of ferulate, we first applied adaptive laboratory evolution to A. baylyi ADP1, resulting in a highly ferulate-tolerant strain. The adapted strain exhibited robust growth in 100 mM ferulate while the growth of the wild type strain was completely inhibited. Next, we expressed two heterologous enzymes in the wild type strain to confer 1-undecene production from glucose: a fatty acid decarboxylase UndA from Pseudomonas putida, and a thioesterase 'TesA from Escherichia coli. Finally, we constructed the 1-undecene synthesis pathway in the ferulate-tolerant strain. The engineered cells were able to produce biomass and 1-undecene solely from ferulate, and excreted the product directly to the culture headspace. CONCLUSIONS: In this study, we employed a bacterium Acinetobacter baylyi ADP1 to integrate a natural aromatics degrading pathway to a synthetic production route, allowing the upgradation of lignin derived molecules to value-added products. We developed a highly ferulate-tolerant strain and established the biosynthesis of an industrially relevant chemical, 1-undecene, solely from the lignin-derived model compound. This study reports the production of alkenes from lignin derived molecules for the first time and demonstrates the potential of lignin as a sustainable resource in the bio-based synthesis of valuable products.

Improved fatty aldehyde and wax ester production by overexpression of fatty acyl-CoA reductases.

BACKGROUND: Fatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different properties are needed for the construction of tailored synthetic pathways for the production of various compounds. RESULTS: Fatty aldehyde production in Acinetobacter baylyi ADP1 was increased by the overexpression of three different FARs: a native A. baylyi FAR Acr1, a cyanobacterial Aar, and a putative, previously uncharacterized dehydrogenase (Ramo) from Nevskia ramosa. The fatty aldehyde production was followed in real-time inside the cells with a luminescence-based tool, and the highest aldehyde production was achieved with Aar. The fate of the overproduced fatty aldehydes was studied by measuring the production of wax esters by a native downstream pathway of A. baylyi, for which fatty aldehyde is a specific intermediate. The wax ester production was improved with the overexpression of Acr1 or Ramo compared to the wild type A. baylyi by more than two-fold, whereas the expression of Aar led to only subtle wax ester production. The overexpression of FARs did not affect the length of the acyl chains of the wax esters. CONCLUSIONS: The fatty aldehyde production, as well as the wax ester production of A. baylyi, was improved with the overexpression of a key enzyme in the pathway. The wax ester titer (0.45 g/l) achieved with the overexpression of Acr1 is the highest reported without hydrocarbon supplementation to the culture. The contrasting behavior of the different reductases highlight the significance of in vivo characterization of enzymes and emphasizes the possibilities provided by the diversity of FARs for pathway and product modulation.

Supraventricular arrhythmias in patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy associate with long-term outcome after catheter ablation of ventricular tachycardias.

Aims: This study aimed to assess the impact of supraventricular tachycardia (SVT) on long-term results of radiofrequency catheter ablation therapy of ventricular tachycardia (VT) in a large cohort of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). Methods and results: Supraventricular tachycardia occurrence has been studied in patients from our ARVD/C registry (70 patients, 48 male, age 53.2 ± 14.0, 45 patients (64.3%) with previous VT ablation). SVT were diagnosed in 26 of 70 patients (37.1%). Atrial fibrillation (AF) was the most frequent atrial arrhythmia, diagnosed in 17 patients (24.3%). In univariate analysis advanced age, clinical symptoms of heart failure, enlarged right atrium, diagnosis of significant tricuspid regurgitation (TR), and inappropriate implantable cardioverters-defibrillators therapy were associated with SVT. In binary logistic regression analysis only heart failure: hazard ratio (HR) 10.89, 95% confidence interval (95% CI) 1.08-109.96 (P = 0.043) and significant TR: HR 4.79, 95% CI 1.35-16.33 (P = 0.015) remained associated with SVT. In patients with previous VT ablation Cox multiple regression survival analysis revealed older age (≥53 years): HR 4.63, 95% CI 1.51-14.24 (P = 0.008) and SVT: HR 3.01, 95% CI 1.15-7.89 (P = 0.025) as predictors for VT recurrence during the follow-up. Conclusion: SVT and older age are associated with the recurrence of VT after catheter ablation in patients with ARVD/C.

Growth and wax ester production of an Acinetobacter baylyi ADP1 mutant deficient in exopolysaccharide capsule synthesis.

Acinetobacter baylyi ADP1 naturally produces wax esters that could be used as a raw material in industrial applications. We attempted to improve wax ester yield of A. baylyi ADP1 by removing rmlA, a gene involved in exopolysaccharide production. Growth rate, biomass formation and wax ester yield on 4-hydroxybenzoate were not affected, but the rmlA - strain grew slower on acetate, while reaching similar biomass and wax ester yield. The rmlA - cells had malformed shape and large size and grew poorly on glucose without expression of the gene for pyruvate kinase (pykF) from Escherichia coli. The pykF-expressing rmlA - strain had similar growth rate, lowered biomass formation and improved wax ester production on glucose as compared to the wild-type strain expressing pykF. Cultivation of the pykF-expressing rmlA - strain on an elevated glucose concentration in a medium supplemented with amino acids resulted in doubled molar wax ester yield and acetate production.

Real-time fluorescence imaging of the DNA damage repair response during mitosis.

The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe.

Comparison of UVB and UVC effects on the DNA damage-response protein 53BP1 in human pancreatic cancer.

We have previously demonstrated that ultraviolet (UV) light is effective against a variety of cancer cells expressing fluorescent proteins in vivo as well as in vitro. In the present report, we compared the DNA damage repair (DDR) response of pancreatic cancer cells after UVB or UVC irradiation. The UV-induced DNA damage repair was imaged with green fluorescent protein (GFP) fused to the DDR-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells growing in 3D Gelfoam® histoculture and in superficial tumors grown in nude mice. 53BP1-GFP forms foci during DNA damage repair. A clonogenic assay in 2D monolayer culture initially showed that UVC and UVB inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, with UVC having more efficacy. Three-dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP foci to be observed within 1 h after UV irradiation, indicating the onset of DDR response. UVB-induced 53BP1-GFP focus formation was observed up to a depth of 120 µm in MiaPaCa-2 cells on Gelfoam® compared to 80 µm for UVC. UVB-induced 53BP1-GFP focus formation was observed up to a depth of 80 µm in MiaPaCa-2 cells, implanted within skin flaps in mice, at a significantly greater extent than UVC. MiaPaCa-2 cells irradiated by UVB or UVC in the skin-flap mouse model had a significant decrease in tumor growth compared to untreated controls with UVB having more efficacy than UVC. Our results demonstrate that UVB has greater tissue penetration than UVC because of its longer wavelength and has clinical potential for eradicating superficial cancer.

Carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria.

BACKGROUND: Lignin is a highly abundant but strongly underutilized natural resource that could serve as a sustainable feedstock for producing chemicals by microbial cell factories. Because of the heterogeneous nature of the lignin feedstocks, the biological upgrading of lignin relying on the metabolic routes of aerobic bacteria is currently considered as the most promising approach. However, the limited substrate range and the inefficient catabolism of the production hosts hinder the upgrading of lignin-related aromatics. Particularly, the aerobic O-demethylation of the methoxyl groups in aromatic substrates is energy-limited, inhibits growth, and results in carbon loss in the form of CO2. RESULTS: In this study, we present a novel approach for carbon-wise utilization of lignin-related aromatics by the integration of anaerobic and aerobic metabolisms. In practice, we employed an acetogenic bacterium Acetobacterium woodii for anaerobic O-demethylation of aromatic compounds, which distinctively differs from the aerobic O-demethylation; in the process, the carbon from the methoxyl groups is fixed together with CO2 to form acetate, while the aromatic ring remains unchanged. These accessible end-metabolites were then utilized by an aerobic bacterium Acinetobacter baylyi ADP1. By utilizing this cocultivation approach, we demonstrated an upgrading of guaiacol, an abundant but inaccessible substrate to most microbes, into a plastic precursor muconate, with a nearly equimolar yields (0.9 mol/mol in a small-scale cultivation and 1.0 mol/mol in a one-pot bioreactor cultivation). The process required only a minor genetic engineering, namely a single gene knock-out. Noticeably, by employing a metabolic integration of the two bacteria, it was possible to produce biomass and muconate by utilizing only CO2 and guaiacol as carbon sources. CONCLUSIONS: By the novel approach, we were able to overcome the issues related to aerobic O-demethylation of methoxylated aromatic substrates and demonstrated carbon-wise conversion of lignin-related aromatics to products with yields unattainable by aerobic processes. This study highlights the power of synergistic integration of distinctive metabolic features of bacteria, thus unlocking new opportunities for harnessing microbial cocultures in upgrading challenging feedstocks.

Enhanced upgrading of lignocellulosic substrates by coculture of Saccharomyces cerevisiae and Acinetobacter baylyi ADP1.

BACKGROUND: Lignocellulosic biomass as feedstock has a huge potential for biochemical production. Still, efficient utilization of hydrolysates derived from lignocellulose is challenged by their complex and heterogeneous composition and the presence of inhibitory compounds, such as furan aldehydes. Using microbial consortia where two specialized microbes complement each other could serve as a potential approach to improve the efficiency of lignocellulosic biomass upgrading. RESULTS: This study describes the simultaneous inhibitor detoxification and production of lactic acid and wax esters from a synthetic lignocellulosic hydrolysate by a defined coculture of engineered Saccharomyces cerevisiae and Acinetobacter baylyi ADP1. A. baylyi ADP1 showed efficient bioconversion of furan aldehydes present in the hydrolysate, namely furfural and 5-hydroxymethylfurfural, and did not compete for substrates with S. cerevisiae, highlighting its potential as a coculture partner. Furthermore, the remaining carbon sources and byproducts of S. cerevisiae were directed to wax ester production by A. baylyi ADP1. The lactic acid productivity of S. cerevisiae was improved approximately 1.5-fold (to 0.41 ± 0.08 g/L/h) in the coculture with A. baylyi ADP1, compared to a monoculture of S. cerevisiae. CONCLUSION: The coculture of yeast and bacterium was shown to improve the consumption of lignocellulosic substrates and the productivity of lactic acid from a synthetic lignocellulosic hydrolysate. The high detoxification capacity and the ability to produce high-value products by A. baylyi ADP1 demonstrates the strain to be a potential candidate for coculture to increase production efficiency and economics of S. cerevisiae fermentations.

Imaging UVC-induced DNA damage response in models of minimal cancer.

We have previously demonstrated that the ultraviolet (UV) light is effective against a variety of cancer cells in vivo as well as in vitro. In the present report, we imaged the DNA damage repair response of minimal cancer after UVC irradiation. DNA-damage repair response to UV irradiation was imaged on tumors growing in 3D culture and in superficial tumors grown in vivo. UV-induced DNA damage repair was imaged with GFP fused to the DNA damage response (DDR)-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells. Three-dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP nuclear foci to be observed within 1 h after UVC irradiation, indicating the onset of DNA damage repair response. A clonogenic assay showed that UVC inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, while UVA and UVB showed little effect on cell proliferation. Induction of UV-induced 53BP1-GFP focus formation was limited up to a depth of 40 µm in 3D-culture of MiaPaCa-2 cells. The MiaPaCa-2 cells irradiated by UVC light in a skin-flap mouse model had a significant decrease of tumor growth compared to untreated controls. Our results also demonstrate that 53BP1-GFP is an imageable marker of UV-induced DNA damage repair response of minimal cancer and that UVC is a useful tool for the treatment of residual cancer since UVC can kill superficial cancer cells without damage to deep tissue.

Dynamic color-coded fluorescence imaging of the cell-cycle phase, mitosis, and apoptosis demonstrates how caffeine modulates cisplatinum efficacy.

Caffeine enhances the effect of certain anticancer drugs, but the mechanism of modulation is poorly understood. In this study, modulation of cisplatinum efficacy induced by caffeine was visualized at the subcellular level by real-time fluorescent-protein imaging. Mitotic and apoptotic changes were observed by imaging 143B human osteosarcoma dual-color cells, in which GFP is expressed in the nucleus and RFP is expressed in the cytoplasm. Modulation of the cell cycle was imaged using time-lapse imaging of HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (FUCCI) in the nucleus. Clonogenic assays showed that caffeine increased the inhibition by cisplatinum on cell proliferation. Subcellular imaging demonstrated that cisplatinum decreased mitosis and induced apoptosis in 143B cells. The combination of cisplatinum and caffeine enhanced mitosis and subsequently increased apoptosis. Time-lapse imaging showed that cisplatinum strongly induced cell-cycle arrest in the S/G2 phase in HeLa-FUCCI cells. Caffeine overcame the cell-cycle arrest induced by cisplatinum, thereby increasing its efficacy, since cisplatinum is ineffective against quiescent cells. The data in this report indicate that caffeine modulates the cell cycle in cancer cells, thereby enhancing efficacy of cell-cycle-dependent anticancer drugs such as cisplatinum.

Left ventricular function improvement after prophylactic implantable cardioverter-defibrillator implantation in patients with non-ischaemic dilated cardiomyopathy.

AIMS: To assess the incidence and prognostic significance of left ventricular (LV) function improvement in patients with non-ischaemic dilated cardiomyopathy (DCM) and prophylactic implantable cardioverter-defibrillator (ICD). METHODS AND RESULTS: A total of 123 patients with DCM and echocardiographic follow-up assessments within 1 year after prophylactic ICD implant were retrospectively studied at our institution. All patients had New York Heart Association class II or III symptoms in the presence of a LV ejection fraction of 23 ± 6% (range: 9-35%) despite optimized medical therapy for at least 3 months prior to ICD implant. Left ventricular function improvement was defined as an increase of LV ejection fraction of more than 5% to more than 35% combined with a decrease LV end-diastolic diameter of at least 5 mm. Left ventricular function improvement after prophylactic ICD implant was found in 30 of 123 patients (24%). Multivariate logistic regression revealed recent onset DCM with symptoms ≤9 months as the only significant predictor of LV function improvement [odds ratio: 6.89; 95% confidence interval (CI): 2.43-21.99, P = 0.0002]. During 74 months mean follow-up, total mortality was higher in patients without vs. with LV function improvement [hazard ratio (HR): 3.75; 95% CI: 1.14-12.31, P = 0.0034], while the incidence of appropriate ICD therapies was similar in both groups in the early phase after prophylactic ICD implant (HR: 1.15; 95% CI: 0.57-2.33, P = 0.70). The incidence of appropriate ICD therapies decreased to ∼1% per year after LV function improvement had occurred. CONCLUSION: Recently diagnosed DCM predicts LV function improvement after prophylactic ICD implant. Overall survival was significantly better in patients with vs. without LV function improvement, while appropriate ICD therapy rates were similar in both groups in the early phase after prophylactic ICD implantation before LV function improvement occurred.

Radiation-inducible immunotherapy for cancer: senescent tumor cells as a cancer vaccine.

Radiotherapy offers an effective treatment for advanced cancer but local and distant failures remain a significant challenge. Here, we treated melanoma and pancreatic carcinoma in syngeneic mice with ionizing radiation (IR) combined with the poly(ADP-ribose) polymerase inhibitor (PARPi) veliparib to inhibit DNA repair and promote accelerated senescence. Based on prior work implicating cytotoxic T lymphocytes (CTLs) as key mediators of radiation effects, we discovered that senescent tumor cells induced by radiation and veliparib express immunostimulatory cytokines to activate CTLs that mediate an effective antitumor response. When these senescent tumor cells were injected into tumor-bearing mice, an antitumor CTL response was induced which potentiated the effects of radiation, resulting in elimination of established tumors. Applied to human cancers, radiation-inducible immunotherapy may enhance radiotherapy responses to prevent local recurrence and distant metastasis.

Prescribed Versus Taken Polypharmacy and Drug-Drug Interactions in Older Cardiovascular Patients during the COVID-19 Pandemic: Observational Cross-Sectional Analytical Study.

The study aimed to assess clinical pharmacology patterns of prescribed and taken medications in older cardiovascular patients using electronic health records (EHRs) (n = 704) (2019-2022). Medscape Drug Interaction Checker was used to identify pairwise drug-drug interactions (DDIs). Prevalence rates of DDIs were 73.5% and 68.5% among taken and prescribed drugs, respectively. However, the total number of DDIs was significantly higher among the prescribed medications (p < 0.05). Serious DDIs comprised 16% and 7% of all DDIs among the prescribed and taken medications, respectively (p < 0.05). Median numbers of DDIs between the prescribed vs. taken medications were Me = 2, IQR 0-7 vs. Me = 3, IQR 0-7 per record, respectively. Prevalence of polypharmacy was significantly higher among the prescribed medications compared with that among the taken drugs (p < 0.05). Women were taking significantly more drugs and had higher prevalence of polypharmacy and DDIs (p < 0.05). No sex-related differences were observed in the list of prescribed medications. ICD code U07.1 (COVID-19, virus identified) was associated with the highest median DDI number per record. Further research is warranted to improve EHR structure, implement patient engagement in reporting adverse drug reactions, and provide genetic profiling of patients to avoid potentially serious DDIs.

Engineering cell morphology by CRISPR interference in Acinetobacter baylyi ADP1.

Microbial production of intracellular compounds can be engineered by redirecting the carbon flux towards products and increasing the cell size. Potential engineering strategies include exploiting clustered regularly interspaced short palindromic repeats interference (CRISPRi)-based tools for controlling gene expression. Here, we applied CRISPRi for engineering Acinetobacter baylyi ADP1, a model bacterium for synthesizing intracellular storage lipids, namely wax esters. We first established an inducible CRISPRi system for strain ADP1, which enables tightly controlled repression of target genes. We then targeted the glyoxylate shunt to redirect carbon flow towards wax esters. Second, we successfully employed CRISPRi for modifying cell morphology by repressing ftsZ, an essential gene required for cell division, in combination with targeted knock-outs to generate significantly enlarged filamentous or spherical cells respectively. The engineered cells sustained increased wax ester production metrics, demonstrating the potential of cell morphology engineering in the production of intracellular lipids.

Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization.

Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT's non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT's role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.

Progression of cancer from indolent to aggressive despite antigen retention and increased expression of interferon-gamma inducible genes.

Many cancers escape host immunity without losing tumor-specific rejection antigens or MHC class I expression. This study tracks the evolution of one such cancer that developed in a mouse following exposure to ultraviolet light. The primary autochthonous tumor was not highly malignant and was rejected when transplanted into naïve immunocompetent mice. Neoplastic cells isolated from the primary tumor were susceptible to the growth-inhibitory effects of IFNγ in vitro, but expressed very low levels of MHC I antigen and were resistant to tumor-specific T cells unless they were first exposed to IFNγ. Serial passage of the primary tumor cells in vivo led to a highly aggressive variant that caused fast-growing tumors in normal mice. In vitro, the variant tumor cells showed increased resistance to the growth-inhibitory effects of IFNγ but expressed high levels of immunoproteasomes and MHC I molecules and were susceptible to tumor-specific T cells even without prior exposure to IFNγ.

Real-time monitoring of intracellular wax ester metabolism.

BACKGROUND: Wax esters are industrially relevant molecules exploited in several applications of oleochemistry and food industry. At the moment, the production processes mostly rely on chemical synthesis from rather expensive starting materials, and therefore solutions are sought from biotechnology. Bacterial wax esters are attractive alternatives, and especially the wax ester metabolism of Acinetobacter sp. has been extensively studied. However, the lack of suitable tools for rapid and simple monitoring of wax ester metabolism in vivo has partly restricted the screening and analyses of potential hosts and optimal conditions. RESULTS: Based on sensitive and specific detection of intracellular long-chain aldehydes, specific intermediates of wax ester synthesis, bacterial luciferase (LuxAB) was exploited in studying the wax ester metabolism in Acinetobacter baylyi ADP1. Luminescence was detected in the cultivation of the strain producing wax esters, and the changes in signal levels could be linked to corresponding cell growth and wax ester synthesis phases. CONCLUSIONS: The monitoring system showed correlation between wax ester synthesis pattern and luminescent signal. The system shows potential for real-time screening purposes and studies on bacterial wax esters, revealing new aspects to dynamics and role of wax ester metabolism in bacteria.