mTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum.

Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans.

CCM3 Loss-Induced Lymphatic Defect Is Mediated by the Augmented VEGFR3-ERK1/2 Signaling.

OBJECTIVE: Cerebral cavernous malformations (CCMs) can happen anywhere in the body, although they most commonly produce symptoms in the brain. The role of CCM genes in other vascular beds outside the brain and retina is not well-examined, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in lymphatics. Approach and Results: Mice with an inducible pan-endothelial cell (EC) or lymphatic EC deletion of Ccm3 (Pdcd10ECKO or Pdcd10LECKO) exhibit dilated lymphatic capillaries and collecting vessels with abnormal valve structure. Morphological alterations were correlated with lymphatic dysfunction in Pdcd10LECKO mice as determined by Evans blue dye and fluorescein isothiocyanate(FITC)-dextran transport assays. Pdcd10LECKO lymphatics had increased VEGFR3 (vascular endothelial growth factor receptor-3)-ERK1/2 (extracellular signal-regulated kinase 1/2) signaling with lymphatic hyperplasia. Mechanistic studies suggested that VEGFR3 is primarily regulated at a transcriptional level in Ccm3-deficient lymphatic ECs, in an NF-κB (nuclear factor κB)-dependent manner. CCM3 binds to importin alpha 2/KPNA2 (karyopherin subunit alpha 2), and a CCM3 deletion releases KPNA2 to activate NF-κB P65 by facilitating its nuclear translocation and P65-dependent VEGFR3 transcription. Moreover, increased VEGFR3 in lymphatic EC preferentially activates ERK1/2 signaling, which is critical for lymphatic EC proliferation. Importantly, inhibition of VEGFR3 or ERK1/2 rescued the lymphatic defects in structure and function. CONCLUSIONS: Our data demonstrate that CCM3 deletion augments the VEGFR3-ERK1/2 signaling in lymphatic EC that drives lymphatic hyperplasia and malformation and warrant further investigation on the potential clinical relevance of lymphatic dysfunction in patients with CCM.

Bleeding with iron deposition and vascular remodelling in subchondral cysts: A newly discovered feature unique to haemophilic arthropathy.

INTRODUCTION: Joint iron accumulation is the incendiary factor triggering osteochondral destruction, synovial hypertrophy, inflammation, and vascular remodelling in haemophilic arthropathy (HA). Hemosiderin depositions have been described in synovium and, more recently, in cartilage. Clinical observations also suggest hemosiderin accumulation in subchondral cysts, implying cyst bleeding. AIM: We explored associations between cystic iron accumulation, vascular remodelling and HA status to determine if cystic bleeding may contribute to HA progression. METHODS: Thirty-six haemophilic joints (16 knees, 10 ankles, and 10 elbows; 31 adult patients with haemophilia A/B) were evaluated by magnetic resonance imaging (MRI) for subchondral cysts and hemosiderin. Cyst score (WORMS) and hemosiderin presence were compared between haemophilic and osteoarthritic knees, matched for the degree of arthritis (Kellgren-Lawrence score). Cystic iron accumulation, vascular remodelling and macrophage cell counts were also compared by immunohistochemistry in explanted joint tissues. In haemophilic knees, cyst number and extent of hemosiderin deposition were correlated with haemophilia joint health scores (HJHS). RESULTS: Cystic hemosiderin was detected in 78% of haemophilic joints. Cyst score and presence of hemosiderin were significantly higher in haemophilic compared to osteoarthritic knees. Cyst score and presence of hemosiderin strongly correlated with HJHS. Moreover, iron deposition and vascular remodelling were significantly more pronounced within cysts in haemophilic compared to osteoarthritic knees, with similar total cell and macrophage count. CONCLUSION: These findings suggest the presence of subchondral bleeding in haemophilia, contributing to poor joint health outcomes. Observations of bleeding into osseous structures are novel and should inform investigations of new therapies.

Conversion of viridicatic acid to crustosic acid by cytochrome P450 enzyme-catalysed hydroxylation and spontaneous cyclisation.

Cytochrome P450 monooxygenases (P450s) are considered nature's most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. KEY POINTS: •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

POEMS (POLYMERIC OPTO-ELECTRO-MECHANICAL SYSTEMS) FOR ADVANCED NEURAL INTERFACES.

There has been a growing interest in optical neural interfaces which is driven by the need for improvements in spatial precision, real-time monitoring, and reduced invasiveness. Here, we present unique microfabrication and packaging techniques to build implantable optoelectronics with high precision and spatial complexity. Material characterization of our hybrid polymers shows minimal in vitro degradation, greater flexibility, and lowest optical loss (4.04-4.4 dB/cm at 670 nm) among other polymers reported in prior studies. We use the developed methods to build Lawrence Livermore National Laboratory's (LLNL's) first ultra-compact, lightweight (0.38 g), scalable and minimally invasive thin-film optoelectronic neural implant that can be used for chronic studies of brain activities. The paper concludes by summarizing the progress to date and discussing future opportunities for flexible optoelectronic interfaces in next generation clinical applications.

Ultrashort echo time quantitative susceptibility mapping (UTE-QSM) for detection of hemosiderin deposition in hemophilic arthropathy: A feasibility study.

PURPOSE: The purpose of this study was to investigate the feasibility of ultrashort echo time quantitative susceptibility mapping (UTE-QSM) for assessment of hemosiderin deposition in the joints of hemophilic patients. METHODS: The UTE-QSM technique was based on three sets of dual-echo 3D UTE Cones data acquired with TEs of 0.032/2.8, 0.2/3.6, and 0.4/4.4 ms. The images were processed with iterative decomposition of water and fat with echo asymmetry and least-squares estimation to estimate the B0 field map in the presence of fat. Then, the projection onto dipole field (PDF) algorithm was applied to acquire a local field map generated by tissues, followed by application of the morphology-enabled dipole inversion (MEDI) algorithm to estimate a final susceptibility map. Three healthy volunteers and three hemophilic patients were recruited to evaluate the UTE-QSM technique's ability to assess hemosiderin in the knee or ankle joint at 3T. One patient subsequently underwent total knee arthroplasty after the MR scan. The synovial tissues harvested from the knee joint during surgery were processed for histological analysis to confirm iron deposition. RESULTS: UTE-QSM successfully yielded tissue susceptibility maps of joints in both volunteers and patients. Multiple regions with high susceptibility over 1 ppm were detected in the affected joints of hemophilic patients, while no localized regions with high susceptibility were detected in asymptomatic healthy volunteers. Histology confirmed the presence of iron in regions where high susceptibility was detected by UTE-QSM. CONCLUSION: The UTE-QSM technique can detect hemosiderin deposition in the joint, and provides a potential sensitive biomarker for the diagnosis and prognosis of hemophilic arthropathy.

Development and Reliability of the Joint Tissue Activity and Damage Examination for Quantitation of Structural Abnormalities by Musculoskeletal Ultrasound in Hemophilic Joints.

OBJECTIVES: Musculoskeletal ultrasound (US) is used increasingly to examine hemophilic arthropathy. However, quantitative algorithms to document findings are lacking. We developed and sought to validate a protocol quantifying hemophilic joint abnormalities. METHODS: Thirty-one patients with hemophilia were examined serially for 2 years with musculoskeletal US (≈600 joint examinations and ≈6000 images). Based on the spectrum of pathologies, a quantitative algorithm, named Joint Tissue Activity and Damage Examination (JADE), was developed for soft tissue and osteochondral measurements, including power Doppler, using nominal group techniques. To study intra- and inter-rater reliability, 8 musculoskeletal US-experienced hemophilia providers performed anatomic landmark recognition and tissue measurements on 86 images with arthropathic changes, with repetition 1 month later. Twenty-three musculoskeletal US-inexperienced providers performed similar assessments. Inter-operator reliability was established by 6 musculoskeletal US-experienced hemophilia providers, each acquiring images and JADE assessments of 3 hemophilic arthropathic joints. A radiologist and musculoskeletal sonographer functioned as adjudicators. The statistical analysis was performed with the intraclass correlation coefficient (ICC), Fleiss κ, and Cohen κ where appropriate. RESULTS: The musculoskeletal US-experienced providers showed excellent intra-and inter-rater reliability for tissue measurements (ICCs, 0.94-0.96). Agreement was good to excellent for landmark recognition (Fleiss κ, 0.87-0.94). Inter-operator reliability was excellent for measurements and landmark recognition (ICC, 0.90; Fleiss κ, 1.0). Agreement with adjudicators was mostly good to excellent. Musculoskeletal US-inexperienced providers showed excellent inter-rater reliability for measurements (ICC, 0.96) and moderate agreement for landmark recognition (Fleiss κ, 0.58). CONCLUSIONS: The JADE protocol appears feasible for quantifying hemophilic intra-articular abnormalities. Musculoskeletal US-trained hemophilia providers showed high intra-rater, inter-rater, and inter-operator reliability, supporting JADE as a protocol for clinical management and research.

Objective response to mTOR inhibition in a metastatic collision tumor of the liver composed of melanoma and adenocarcinoma with TSC1 loss: a case report.

BACKGROUND: Collision tumors are uncommon but well described clinical entities composed of distinct tumor histologies occurring within the same anatomic site. Optimal management of patients with collision tumors remains highly variable and depends on clinical characteristics such as the involved tumor types, predominant histology, as well as the extent of disease. Comprehensive genomic profiling is a means of identifying genomic alterations to suggest benefit from targeted therapy. CASE PRESENTATION: A 78-year-old woman presented to medical oncology with liver metastases occurring within the background of a 1-year history of uveal melanoma. Biopsy of the liver metastases revealed presence of adenocarcinoma along with nests of malignant melanoma consistent with a collision tumor. The disease was refractory to several lines of conventional cytotoxic chemotherapy, and the patient later developed pulmonary metastases while on chemotherapy. The patient's tumor tissue was assayed by comprehensive genomic profiling which revealed presence of a TSC1 partial loss. The patient was subsequently initiated on temsirolimus 15 mg intravenously weekly for 4 months. Restaging imaging demonstrated a partial response to therapy by RECIST 1.1 criteria and clinical benefit for 6 months until the patient passed away secondary to unrelated causes. CONCLUSIONS: We report the first case of a collision tumor composed of adenocarcinoma and melanoma with a TSC1 mutation that objectively and durably responded to mTOR inhibition.

Regional specialization manifests in the reliability of neural population codes.

The brain has the remarkable ability to learn and guide the performance of complex tasks. Decades of lesion studies suggest that different brain regions perform specialized functions in support of complex behaviors1-3. Yet recent large-scale studies of neural activity reveal similar patterns of activity and encoding distributed widely throughout the brain4-6. How these distributed patterns of activity and encoding are compatible with regional specialization of brain function remains unclear. Two frontal brain regions, the dorsal medial prefrontal cortex (dmPFC) and orbitofrontal cortex (OFC), are a paradigm of this conundrum. In the setting complex behaviors, the dmPFC is necessary for choosing optimal actions2,7,8, whereas the OFC is necessary for waiting for3,9 and learning from2,7,9-12 the outcomes of those actions. Yet both dmPFC and OFC encode both choice- and outcome-related quantities13-20. Here we show that while ensembles of neurons in the dmPFC and OFC of rats encode similar elements of a cognitive task with similar patterns of activity, the two regions differ in when that coding is consistent across trials ("reliable"). In line with the known critical functions of each region, dmPFC activity is more reliable when animals are making choices and less reliable preceding outcomes, whereas OFC activity shows the opposite pattern. Our findings identify the dynamic reliability of neural population codes as a mechanism whereby different brain regions may support distinct cognitive functions despite exhibiting similar patterns of activity and encoding similar quantities.

Endothelial Cell-Pericyte Interactions in the Pathogenesis of Cerebral Cavernous Malformations (CCMs).

Cerebral cavernous malformations (CCMs), consisting of multiple, dilated capillary channels formed by a single layer of endothelium and lacking parenchymal cells, are exclusively to the brain. Patients with inherited autosomal-dominant CCMs carry loss-of-function mutations in one of three genes: CCM1, CCM2, and CCM3. It is not known why CCM lesions are confined to brain vasculature despite the ubiquitous expression of CCM proteins in all tissues, and whether cell types other than endothelial cells (ECs) contribute to CCM lesion formation. The prevailing view is that the primary defects in CCMs in humans are EC-intrinsic, such that EC-specific deletion of any one of the three genes in mice results in similar CCM lesions. An unexpected finding is that Ccm3 deletion in pericytes (PCs) also induces CCM lesions. CCM3 deletion in ECs or PCs destabilizes PC-EC associations, highlighting the importance of these interactions in CCM formation.

Association between mental health and self-care behavior among older adults with diabetes according to Behavioral Risk Factor Surveillance System 2019.

AIM: To assess the association of mental health burden with diabetes-related self-care behaviors and healthcare utilization among older adults. METHOD: This cross-sectional 2019 Behavioral Risk Factor Surveillance System (BRFSS) study included ≥ 65 years old adults with self-reported diabetes. Three groups were used based on the number of days in the past month affected by mental health: 0 days (no burden), 1-13 days (occasional burden), and 14-30 days (frequent burden). Primary outcome was performing ≥ 3 of 5 diabetes-related self-care behaviors. Secondary outcome was performing ≥ 3 of 5 healthcare utilization behaviors. Multivariable logistic regression was used in Stata/SE 15.1. RESULTS: Of 14,217 included individuals, 10.2 % reported frequent mental health burden. Compared to 'no burden', 'occasional' and 'frequent burden' groups included more female, obese, not married persons with younger age of diabetes diagnosis, and reported more comorbidities, insulin use, cost-related barriers to see doctors, and diabetes-related eye issues (p < 0.05). 'Occasional/frequent burden' groups reported less self-care and healthcare utilization behaviors, except 30 % higher healthcare utilization was observed in the 'occasional burden' group compared to no burden (aOR 1.30, 95 %CI 1.08-1.58, p = 0.006). CONCLUSIONS: Overall, mental health burden was associated with reduced participation in diabetes-related self-care and healthcare utilization behaviors in a stepwise manner, except occasional burden was associated with higher healthcare utilization.

SRF SUMOylation modulates smooth muscle phenotypic switch and vascular remodeling.

Serum response factor (SRF) controls gene transcription in vascular smooth muscle cells (VSMCs) and regulates VSMC phenotypic switch from a contractile to a synthetic state, which plays a key role in the pathogenesis of cardiovascular diseases (CVD). SRF activity is regulated by its associated cofactors. However, it is not known how post-translational SUMOylation regulates the SRF activity in CVD. Here, we show that Senp1 deficiency in VSMCs increased SUMOylated SRF and the SRF-ELK complex, leading to augmented vascular remodeling and neointimal formation in mice. Mechanistically, SENP1 deficiency in VSMCs increased SRF SUMOylation at lysine 143, which reduced its lysosomal localization concomitant with increased nuclear accumulation. SUMOylation of SRF switched its binding with the contractile phenotype-responsive cofactor myocardin to binding with the synthetic phenotype-responsive cofactor phosphorylated ELK1. Both SUMOylated SRF and phosphor-ELK1 were increased in VSMCs from coronary arteries of CVD patients. Importantly, preventing the shift from SRF-myocardin to SRF-ELK complex by AZD6244 inhibited the excessive proliferative, migratory, and synthetic phenotypes, attenuating neointimal formation in Senp1-deficient mice. Therefore, targeting the SRF complex may have a therapeutic potential for the treatment of CVD.

Spread of activation and interaction between channels with multi-channel optogenetic stimulation in the mouse cochlea.

For individuals with severe to profound hearing loss resulting from irreversibly damaged hair cells, cochlear implants can be used to restore hearing by delivering electrical stimulation directly to the spiral ganglion neurons. However, current spread lowers the spatial resolution of neural activation. Since light can be easily confined, optogenetics is a technique that has the potential to improve the precision of neural activation, whereby visible light is used to stimulate neurons that are modified with light-sensitive opsins. This study compares the spread of neural activity across the inferior colliculus of the auditory midbrain during electrical and optical stimulation in the cochlea of acutely deafened mice with opsin-modified spiral ganglion neurons (H134R variant of the channelrhodopsin-2). Monopolar electrical stimulation was delivered via each of four 0.2 mm wide platinum electrode rings at 0.6 mm centre-to-centre spacing, whereas 453 nm wavelength light was delivered via each of five 0.22 × 0.27 mm micro-light emitting diodes (LEDs) at 0.52 mm centre-to-centre spacing. Channel interactions were also quantified by threshold changes during simultaneous stimulation by pairs of electrodes or micro-LEDs at different distances between the electrodes (0.6, 1.2 and 1.8 mm) or micro-LEDs (0.52, 1.04, 1.56 and 2.08 mm). The spread of activation resulting from single channel optical stimulation was approximately half that of monopolar electrical stimulation as measured at two levels of discrimination above threshold (p<0.001), whereas there was no significant difference between optical stimulation in opsin-modified deafened mice and pure tone acoustic stimulation in normal-hearing mice. During simultaneous micro-LED stimulation, there were minimal channel interactions for all micro-LED spacings tested. For neighbouring micro-LEDs/electrodes, the relative influence on threshold was 13-fold less for optical stimulation compared electrical stimulation (p<0.05). The outcomes of this study show that the higher spatial precision of optogenetic stimulation results in reduced channel interaction compared to electrical stimulation, which could increase the number of independent channels in a cochlear implant. Increased spatial resolution and the ability to activate more than one channel simultaneously could lead to better speech perception in cochlear implant recipients.

Five Challenging Cases of Hereditary Antithrombin Deficiency Characterized by Thrombosis or Complicated Pregnancy.

Hereditary antithrombin deficiency (ATD) is a rare autosomal dominant condition (estimated prevalence 1:500-1:5000). Most ATD patients have AT activity levels 40-60% of normal. We present treatments for venous thromboembolism (VTE) in five cases of hereditary ATD. Four patients had a family history of ATD, and one had a de novo mutation. The majority of patients had a VTE while on prophylactic anticoagulation. AT concentrate augmentation was added in these cases to treat the VTE and for prophylaxis against further episodes. Two patients had significant bleeding events, one had permanent physical sequelae. Two of the patients were pregnant. VTE is a common cause of morbidity and mortality during pregnancy. Although low molecular weight heparins are the drugs of choice during pregnancy, this treatment was inadequate in one patient (developed VTE on therapy). These cases emphasize the need to screen for ATD in young patients (<55 years) presenting with VTE. AT augmentation therapy may be necessary in patients inadequately treated with conventional anticoagulants. Careful monitoring and individualized care are needed in ATD patients, especially those with demonstrated bleeding tendencies.

Mitochondrial dysfunction induces ALK5-SMAD2-mediated hypovascularization and arteriovenous malformations in mouse retinas.

Although mitochondrial activity is critical for angiogenesis, its mechanism is not entirely clear. Here we show that mice with endothelial deficiency of any one of the three nuclear genes encoding for mitochondrial proteins, transcriptional factor (TFAM), respiratory complex IV component (COX10), or redox protein thioredoxin 2 (TRX2), exhibit retarded retinal vessel growth and arteriovenous malformations (AVM). Single-cell RNA-seq analyses indicate that retinal ECs from the three mutant mice have increased TGFß signaling and altered gene expressions associated with vascular maturation and extracellular matrix, correlating with vascular malformation and increased basement membrane thickening in microvesels of mutant retinas. Mechanistic studies suggest that mitochondrial dysfunction from Tfam, Cox10, or Trx2 depletion induces a mitochondrial localization and MAPKs-mediated phosphorylation of SMAD2, leading to enhanced ALK5-SMAD2 signaling. Importantly, pharmacological blockade of ALK5 signaling or genetic deficiency of SMAD2 prevented retinal vessel growth retardation and AVM in all three mutant mice. Our studies uncover a novel mechanism whereby mitochondrial dysfunction via the ALK5-SMAD2 signaling induces retinal vascular malformations, and have therapeutic values for the alleviation of angiogenesis-associated human retinal diseases.

Tumor hyaluronan as a novel biomarker in non-small cell lung cancer: A retrospective study.

INTRODUCTION: Hyaluronan (HA) accumulation is associated with tumorigenesis and aggressive tumor behavior. AIMS: We investigated the biomarker potential of HA in non-small cell lung cancer (NSCLC). METHODS: HA levels were scored using affinity histochemistry in 137 NSCLC samples stratified by HA score ≤10, 11-20, 21-30, and >30 with HA-high defined as ≥25% expression in the extracellular matrix (ECM) of the tumor surface area. Overall survival (OS) and time to progression from initiation of taxane therapy (TTP) were compared using log-rank tests based on HA score. RESULTS: Of 122 patients with recurrent/metastatic NSCLC, 93 had mean HA scores that were not significantly different across clinicopathologic variables. Frequency of HA-high tumors did not differ by histology (34/68 adenocarcinomas vs. 12/25 squamous tumors, Fisher's p = 1.0000). Median OS for recurrent/metastatic adenocarcinoma was 35.5 months (95%, 23.6-50.3) vs. 17.9 months for squamous (95%, 12.7-37.0, log-rank test, p = 0.0165). OS was not significantly different by HA quartiles, high or low (<25) HA score and tumor histology, and HA biopsy site (all p > 0.05). Median TTP (n = 98) significantly differed by HA quartile (2.8 months for HA score ≤10; 5.0 months for 11-20; 7.9 months for 21-30; 3.9 months for >30, p = 0.0265). Improved TTP trended in HA-high over HA-low tumors (n = 98, p = 0.0911). CONCLUSION: In this NSCLC cohort, tumor HA level represents a potential biomarker for TTP, which remains a cornerstone of NSCLC therapy. Further validation is warranted to identify the HA accumulation threshold associated with clinical benefit.

Cucumber-Derived Exosome-like Vesicles and PlantCrystals for Improved Dermal Drug Delivery.

(1) Background: Extracellular vesicles (EVs) are considered to be efficient nanocarriers for improved drug delivery and can be derived from mammalian or plant cells. Cucumber-derived EVs are not yet described in the literature. Therefore, the aim of this study was to produce and characterize cucumber-derived EVs and to investigate their suitability to improve the dermal penetration efficacy of a lipophilic active ingredient (AI) surrogate. (2) Methods: The EVs were obtained by classical EVs isolation methods and by high pressure homogenization (HPH). They were characterized regarding their physico-chemical and biopharmaceutical properties. (3) Results: Utilization of classical isolation and purification methods for EVs resulted in cucumber-derived EVs. Their dermal penetration efficacy for the AI surrogate was 2-fold higher when compared to a classical formulation and enabled a pronounced transdermal penetration into the viable dermis. HPH resulted in submicron sized particles composed of a mixture of disrupted plant cells. A successful isolation of pure EVs from this mixture was not possible with classical EVs isolation methods. The presence of EVs was, therefore, proven indirectly. For this, the lipophilic drug surrogate was admixed to the cucumber juice either prior to or after HPH. Admixing of the drug surrogate to the cucumber prior to the HPH resulted in a 1.5-fold increase in the dermal penetration efficacy, whereas the addition of the AI surrogate to the cucumber after HPH was not able to improve the penetration efficacy. (4) Conclusions: Results, therefore, indicate that HPH causes the formation of EVs in which AI can be incorporated. The formation of plant EVs by HPH was also indicated by zeta potential analysis.

A Bioinspired Artificial Injury Response System Based on a Robust Polymer Memristor to Mimic a Sense of Pain, Sign of Injury, and Healing.

Flexible electronic skin with features that include sensing, processing, and responding to stimuli have transformed human-robot interactions. However, more advanced capabilities, such as human-like self-protection modalities with a sense of pain, sign of injury, and healing, are more challenging. Herein, a novel, flexible, and robust diffusive memristor based on a copolymer of chlorotrifluoroethylene and vinylidene fluoride (FK-800) as an artificial nociceptor (pain sensor) is reported. Devices composed of Ag/FK-800/Pt have outstanding switching endurance >106  cycles, orders of magnitude higher than any other two-terminal polymer/organic memristors in literature (typically 102 -103 cycles). In situ conductive atomic force microscopy is employed to dynamically switch individual filaments, which demonstrates that conductive filaments correlate with polymer grain boundaries and FK-800 has superior morphological stability under repeated switching cycles. It is hypothesized that the high thermal stability and high elasticity of FK-800 contribute to the stability under local Joule heating associated with electrical switching. To mimic biological nociceptors, four signature nociceptive characteristics are demonstrated: threshold triggering, no adaptation, relaxation, and sensitization. Lastly, by integrating a triboelectric generator (artificial mechanoreceptor), memristor (artificial nociceptor), and light emitting diode (artificial bruise), the first bioinspired injury response system capable of sensing pain, showing signs of injury, and healing, is demonstrated.

Brown adipose TRX2 deficiency activates mtDNA-NLRP3 to impair thermogenesis and protect against diet-induced insulin resistance.

Brown adipose tissue (BAT), a crucial heat-generating organ, regulates whole-body energy metabolism by mediating thermogenesis. BAT inflammation is implicated in the pathogenesis of mitochondrial dysfunction and impaired thermogenesis. However, the link between BAT inflammation and systematic metabolism remains unclear. Herein, we use mice with BAT deficiency of thioredoxin-2 (TRX2), a protein that scavenges mitochondrial reactive oxygen species (ROS), to evaluate the impact of BAT inflammation on metabolism and thermogenesis and its underlying mechanism. Our results show that BAT-specific TRX2 ablation improves systematic metabolic performance via enhancing lipid uptake, which protects mice from diet-induced obesity, hypertriglyceridemia, and insulin resistance. TRX2 deficiency impairs adaptive thermogenesis by suppressing fatty acid oxidation. Mechanistically, loss of TRX2 induces excessive mitochondrial ROS, mitochondrial integrity disruption, and cytosolic release of mitochondrial DNA, which in turn activate aberrant innate immune responses in BAT, including the cGAS/STING and the NLRP3 inflammasome pathways. We identify NLRP3 as a key converging point, as its inhibition reverses both the thermogenesis defect and the metabolic benefits seen under nutrient overload in BAT-specific Trx2-deficient mice. In conclusion, we identify TRX2 as a critical hub integrating oxidative stress, inflammation, and lipid metabolism in BAT, uncovering an adaptive mechanism underlying the link between BAT inflammation and systematic metabolism.