Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma.

Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8+ T cells were defined by clustering and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8+ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states and demonstrated enhanced antitumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms, and targets for enhancing checkpoint immunotherapy.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

Antitelomerase therapy provokes ALT and mitochondrial adaptive mechanisms in cancer.

To assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm(-/-) mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1ß, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1ß or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy.

Telomerase reactivation following telomere dysfunction yields murine prostate tumors with bone metastases.

To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-ß/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.

Genetic Transformation of Plasmid DNA into Escherichia coli Using High Frequency Electromagnetic Energy.

We present a novel technique of genetic transformation of bacterial cells mediated by high frequency electromagnetic energy (HF EME). Plasmid DNA, pGLO (5.4 kb), was successfully transformed into Escherichia coli JM109 cells after exposure to 18 GHz irradiation at a power density between 5.6 and 30 kW m-2 for 180 s at temperatures ranging from 30 to 40 °C. Transformed bacteria were identified by the expression of green fluorescent protein (GFP) using confocal scanning microscopy (CLSM) and flow cytometry (FC). Approximately 90.7% of HF EME treated viable E. coli cells exhibited uptake of the pGLO plasmid. The interaction of plasmid DNA with bacteria leading to transformation was confirmed by using cryogenic transmission electron microscopy (cryo-TEM). HF EME-induced plasmid DNA transformation was shown to be unique, highly efficient, and cost-effective. HF EME-induced genetic transformation is performed under physiologically friendly conditions in contrast to existing techniques that generate higher temperatures, leading to altered cellular integrity. This technique allows safe delivery of genetic material into bacterial cells, thus providing excellent prospects for applications in microbiome therapeutics and synthetic biology.

Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy.

Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.

Retinoschisin deficiency induces persistent aberrant waves of activity affecting neuroglial signaling in the retina.

Genetic disorders which present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis (XLRS), an early-onset inherited condition caused by mutations in the Rs1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of RS1-deficient male mice, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuro-glial signals as early as postnatal day 15, when eyes normally open. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Müller glia, spanning all retinal layers and disrupting light-induced signaling. This study confers a role to RS1 beyond its function as an adhesion molecule, identifies an early onset for dysfunction in the course of disease, establishing a potential window for disease diagnosis and therapeutic intervention.Significance StatementDevelopmental disorders make it difficult to distinguish pathophysiology due to ongoing disease from pathophysiology due to disrupted development. Here, we investigated a mouse model for X-linked retinoschisis (XLRS), a well-defined monogenic degenerative disease caused by mutations in the Rs1 gene, which codes for the protein retinoschisin. We evaluated the spontaneous activity of explanted retinas lacking retinoschisin at key stages of development using the unbiased approach of ubiquitously expressing GCaMP6f in all retinal neurons, vasculature and glia. In mice lacking RS1, we found an array of novel phenotypes which present around eye-opening, are linked to glutamatergic neurotransmission, and affect visual processing. These data identify novel pathophysiology linked to RS1, and define a window where treatments might be best targeted.

Translocation and fate of nanospheres in pheochromocytoma cells following exposure to synchrotron-sourced terahertz radiation.

The routes by which foreign objects enter cells is well studied; however, their fate following uptake has not been explored extensively. Following exposure to synchrotron-sourced (SS) terahertz (THz) radiation, reversible membrane permeability has been demonstrated in eukaryotic cells by the uptake of nanospheres; nonetheless, cellular localization of the nanospheres remained unclear. This study utilized silica core-shell gold nanospheres (AuSi NS) of diameter 50 ± 5 nm to investigate the fate of nanospheres inside pheochromocytoma (PC 12) cells following SS THz exposure. Fluorescence microscopy was used to confirm nanosphere internalization following 10 min of SS THz exposure in the range 0.5-20 THz. Transmission electron microscopy followed by scanning transmission electron microscopy energy-dispersive spectroscopic (STEM-EDS) analysis was used to confirm the presence of AuSi NS in the cytoplasm or membrane, as single NS or in clusters (22% and 52%, respectively), with the remainder (26%) sequestered in vacuoles. Cellular uptake of NS in response to SS THz radiation could have suitable applications in a vast number of biomedical applications, regenerative medicine, vaccines, cancer therapy, gene and drug delivery.

Power of Infrared and Raman Spectroscopies to Characterize Metal-Organic Frameworks and Investigate Their Interaction with Guest Molecules.

The variety of functionalities and porous structures inherent to metal-organic frameworks (MOFs) together with the facile tunability of their properties makes these materials suitable for a wide range of existing and emerging applications. Many of these applications are based on processes involving interaction of MOFs with guest molecules. To optimize a certain process or successfully design a new one, a thorough knowledge is required about the physicochemical characteristics of materials and the mechanisms of their interaction with guest molecules. To obtain such important information, various complementary analytical techniques are applied, among which vibrational spectroscopy (IR and Raman) plays an important role and is indispensable in many cases. In this review, we critically examine the reported applications of IR and Raman spectroscopies as powerful tools for initial characterization of MOF materials and for studying processes of their interaction with various gases. Both the advantages and the limitations of the technique are considered, and the cases where IR or Raman spectroscopy is preferable are highlighted. Peculiarities of MOFs interaction with specific gases and some inconsistent band assignments are also emphasized. Summarizing the broad analytical possibilities of the IR and Raman spectroscopies, we conclude that it can be applied in combinations with other techniques to explicitly establish the structure, properties, and reactivity of MOFs.

Development of the selective micropulse individual retinal therapy depends on age and type on the Fitzpatrick scale.

PURPOSE: To develop a selective micropulse individual retinal therapy (SMIRT) based on the age and appearance type of the patient, to derive a formula for calculating power, and evaluate clinical efficacy for the treatment of central serous chorioretinopathy (CSCR). METHODS: 73 patients (aged 30-65 years) with acute CSCR and types 1-4 on the Fitzpatrick scale were divided into 2 groups. In the first group (33 patients), the testing of the micropulse mode (50 µs, 2.4%, 10 ms, 100 µm, 0.4-1.9 W) on the Navilas 577 s laser system defined as selective by computer modeling was performed. A logistic regression function based on probability damage detection (PDD) of the 1584 laser spots from power, age, and type on the Fitzpatrick scale was constructed. PDD is the probability of detecting the laser spots using the autofluorescence method. The second group was divided into 4 subgroups of 10 eyes each. Groups 2.1, 2.2, and 2.3 were treated without preliminary testing. The power for Groups 2.1, 2.2, and 2.3 was obtained with the inverse PDD function, so that PDD was 50%, 70%, and 90%, respectively. Control group 2.4 went without treatment. RESULTS: The transmission and absorption coefficients of laser radiation of the eye depend on the age and the Fitzpatrick scale type. In Groups 2.1-2.3, complete resorption of subretinal fluid was observed 3 months after CSCR treatment in 5 (P < 0.35), 8 (P < 0.023), and 10 eyes (P < 0.0008) out of 10, respectively. CONCLUSION: The developed SMIRT is effective for CSCR treatment with PDD 90%.

The multi-faceted mechano-bactericidal mechanism of nanostructured surfaces.

The mechano-bactericidal activity of nanostructured surfaces has become the focus of intensive research toward the development of a new generation of antibacterial surfaces, particularly in the current era of emerging antibiotic resistance. This work demonstrates the effects of an incremental increase of nanopillar height on nanostructure-induced bacterial cell death. We propose that the mechanical lysis of bacterial cells can be influenced by the degree of elasticity and clustering of highly ordered silicon nanopillar arrays. Herein, silicon nanopillar arrays with diameter 35 nm, periodicity 90 nm and increasing heights of 220, 360, and 420 nm were fabricated using deep UV immersion lithography. Nanoarrays of 360-nm-height pillars exhibited the highest degree of bactericidal activity toward both Gram stain-negative Pseudomonas aeruginosa and Gram stain-positive Staphylococcus aureus bacteria, inducing 95 ± 5% and 83 ± 12% cell death, respectively. At heights of 360 nm, increased nanopillar elasticity contributes to the onset of pillar deformation in response to bacterial adhesion to the surface. Theoretical analyses of pillar elasticity confirm that deflection, deformation force, and mechanical energies are more significant for the substrata possessing more flexible pillars. Increased storage and release of mechanical energy may explain the enhanced bactericidal action of these nanopillar arrays toward bacterial cells contacting the surface; however, with further increase of nanopillar height (420 nm), the forces (and tensions) can be partially compensated by irreversible interpillar adhesion that reduces their bactericidal effect. These findings can be used to inform the design of next-generation mechano-responsive surfaces with tuneable bactericidal characteristics for antimicrobial surface technologies.

Programmed Death of Injured Pseudomonas aeruginosa on Mechano-Bactericidal Surfaces.

Mechano-bactericidal surfaces deliver lethal effects to contacting bacteria. Until now, cell death has been attributed to the mechanical stress imparted to the bacterial cell envelope by the surface nanostructures; however, the process of bacterial death encountering nanostructured surfaces has not been fully illuminated. Here, we perform an in-depth investigation of the mechano-bactericidal action of black silicon (bSi) surfaces toward Gram-negative bacteria Pseudomonas aeruginosa. We discover that the mechanical injury is not sufficient to kill the bacteria immediately due to the survival of the inner plasma membrane. Instead, such sublethal mechanical injury leads to apoptosis-like death (ALD) in affected bacteria. In addition, when the mechanical stress is removed, the self-accumulated reactive oxygen species (ROS) incur poststress ALD in damaged cells in a nonstressed environment, revealing that the mechano-bactericidal actions have sustained physiological effects on the bacterium. This work creates a new facet and can introduce many new regulation tools to this field.

Biomimetic Nanopillar Silicon Surfaces Rupture Fungal Spores.

The mechano-bactericidal action of nanostructured surfaces is well-documented; however, synthetic nanostructured surfaces have not yet been explored for their antifungal properties toward filamentous fungal species. In this study, we developed a biomimetic nanostructured surface inspired by dragonfly wings. A high-aspect-ratio nanopillar topography was created on silicon (nano-Si) surfaces using inductively coupled plasma reactive ion etching (ICP RIE). To mimic the superhydrophobic nature of insect wings, the nano-Si was further functionalised with trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFTS). The viability of Aspergillus brasiliensis spores, in contact with either hydrophobic or hydrophilic nano-Si surfaces, was determined using a combination of standard microbiological assays, confocal laser scanning microscopy (CLSM), and focused ion beam scanning electron microscopy (FIB-SEM). Results indicated the breakdown of the fungal spore membrane upon contact with the hydrophilic nano-Si surfaces. By contrast, hydrophobised nano-Si surfaces prevented the initial attachment of the fungal conidia. Hydrophilic nano-Si surfaces exhibited both antifungal and fungicidal properties toward attached A. brasisiensis spores via a 4-fold reduction of attached spores and approximately 9-fold reduction of viable conidia from initial solution after 24 h compared to their planar Si counterparts. Thus, we reveal, for the first time, the physical rupturing of attaching fungal spores by biomimetic hydrophilic nanostructured surfaces.

Electrophoretically Co-Deposited Collagen-Lactoferrin Membranes with Enhanced Pro-Regenerative Properties for Oral Soft Tissue Regeneration.

The quality of soft tissue defect regeneration after dental surgeries largely determines their final success. Collagen membranes have been proposed for the healing of such defects, but in some cases, they do not guarantee a sufficient volume of the regenerated tissue and vascularization. For this purpose, lactoferrin, a protein with natural pro-regenerative, anti-inflammatory, and pro-angiogenic activity, can be added to collagen. In this article, we used a semipermeable barrier-assisted electrophoretic deposition (SBA-EPD) method for the production of collagen-lactoferrin membranes. The membrane structure was studied by SEM, and its mechanical properties were shown. The lactoferrin release kinetics were shown by ELISA within 75 h. When tested in vitro, we demonstrated that the collagen-lactoferrin membranes significantly increased the proliferation of keratinocytes (HaCaT) and fibroblasts (977hTERT) compared to blank collagen membranes. In vivo, on the vestibuloplasty and free gingival graft harvesting models, we showed that collagen-lactoferrin membranes decreased the wound inflammation and increased the healing rates and regeneration quality. In some parameters, collagen-lactoferrin membranes outperformed not only blank collagen membranes, but also the commercial membrane Mucograft®. Thus, we proved that collagen-lactoferrin membranes produced by the SBA-EPD method may be a valuable alternative to commercially used membranes for soft tissue regeneration in the oral cavity.

Assessment of the consumptive safety of mercury in fish from the surface waters of the Vologda region in northwestern Russia.

It is known that the consumption of fish is the main source of mercury intake in human beings. In this study, the concentration of mercury in the muscle tissue of fish from different reservoirs of northwestern Russia was found to be 0.01-1.68 µg/g wet weight. The features of mercury accumulation in the muscle tissue of fish, depending on their type, trophic specialization, body weight, length, and the type of water body, were also revealed. Of the fish studied, 7% had mercury concentrations above the regulatory levels of the Russian Federation. The proportion of examined fish, the consumption of which will lead to an excess of the permissible weekly intake of mercury in the individual, is 44% for preschool children (2-5 years old), 34% for children of primary school age (6-10 years old), and 17% for adults. Special attention is drawn to the fact that the mercury content in fish that does not exceed the sanitary and hygienic standards (normative levels) of the Russian Federation may still be unsafe for the health of the population, especially children.

Combined PARP and HSP90 inhibition: preclinical and Phase 1 evaluation in patients with advanced solid tumours.

PURPOSE: PARP inhibitor resistance may be overcome by combinatorial strategies with agents that disrupt homologous recombination repair (HRR). Multiple HRR pathway components are HSP90 clients, so that HSP90 inhibition leads to abrogation of HRR and sensitisation to PARP inhibition. We performed in vivo preclinical studies of the HSP90 inhibitor onalespib with olaparib and conducted a Phase 1 combination study. PATIENTS AND METHODS: Tolerability and efficacy studies were performed in patient-derived xenograft(PDX) models of ovarian cancer. Clinical safety, tolerability, steady-state pharmacokinetics and preliminary efficacy of olaparib and onalespib were evaluated using a standard 3 + 3 dose-escalation design. RESULTS: Olaparib/onalespib exhibited anti-tumour activity against BRCA1-mutated PDX models with acquired PARPi resistance and PDX models with RB-pathway alterations(CDKN2A loss and CCNE1 overexpression). Phase 1 evaluation revealed that dose levels up to olaparib 300 mg/onalespib 40 mg and olaparib 200 mg/onalespib 80 mg were safe without dose-limiting toxicities. Coadministration of olaparib and onalespib did not appear to affect the steady-state pharmacokinetics of either agent. There were no objective responses, but disease stabilisation ≥24 weeks was observed in 7/22 (32%) evaluable patients including patients with BRCA-mutated ovarian cancers and acquired PARPi resistance and patients with tumours harbouring RB-pathway alterations. CONCLUSIONS: Combining onalespib and olaparib was feasible and demonstrated preliminary evidence of anti-tumour activity.

How Various Drug Delivery Methods Could Aid in the Translation of Genome Prime Editing Technologies.

Drug delivery systems can be engineered to enhance the localization of therapeutics in specific tissues in response to externally applied stimuli and/or local environmental changes. In recent decades, efforts to improve drug delivery techniques at both nano- and macroscale have led to a new era of therapeutic efficacy. Such technological advancements resulted in improved drug delivery systems regularly entering the clinical setting. However, these delivery innovations are unfortunately not always readily applied to newly developed technologies. One of these new and exciting technologies that has been overlooked by drug delivery scientists is prime editing. Prime editing is a novel genome editing technology that exhibits the plug-and-play capability of CRISPR/Cas9 editors while avoiding double-strand DNA breaks throughout the entire process. This article focuses on describing the potential advantages and disadvantages of selecting nanomedicine technologies along with prime editing capabilities for the delivery of cargo.