Exome Sequencing and the Identification of New Genes and Shared Mechanisms in Polymicrogyria.

Importance: Polymicrogyria is the most commonly diagnosed cortical malformation and is associated with neurodevelopmental sequelae including epilepsy, motor abnormalities, and cognitive deficits. Polymicrogyria frequently co-occurs with other brain malformations or as part of syndromic diseases. Past studies of polymicrogyria have defined heterogeneous genetic and nongenetic causes but have explained only a small fraction of cases. Objective: To survey germline genetic causes of polymicrogyria in a large cohort and to consider novel polymicrogyria gene associations. Design, Setting, and Participants: This genetic association study analyzed panel sequencing and exome sequencing of accrued DNA samples from a retrospective cohort of families with members with polymicrogyria. Samples were accrued over more than 20 years (1994 to 2020), and sequencing occurred in 2 stages: panel sequencing (June 2015 to January 2016) and whole-exome sequencing (September 2019 to March 2020). Individuals seen at multiple clinical sites for neurological complaints found to have polymicrogyria on neuroimaging, then referred to the research team by evaluating clinicians, were included in the study. Targeted next-generation sequencing and/or exome sequencing were performed on probands (and available parents and siblings) from 284 families with individuals who had isolated polymicrogyria or polymicrogyria as part of a clinical syndrome and no genetic diagnosis at time of referral from clinic, with sequencing from 275 families passing quality control. Main Outcomes and Measures: The number of families in whom genetic sequencing yielded a molecular diagnosis that explained the polymicrogyria in the family. Secondarily, the relative frequency of different genetic causes of polymicrogyria and whether specific genetic causes were associated with co-occurring head size changes were also analyzed. Results: In 32.7% (90 of 275) of polymicrogyria-affected families, genetic variants were identified that provided satisfactory molecular explanations. Known genes most frequently implicated by polymicrogyria-associated variants in this cohort were PIK3R2, TUBB2B, COL4A1, and SCN3A. Six candidate novel polymicrogyria genes were identified or confirmed: de novo missense variants in PANX1, QRICH1, and SCN2A and compound heterozygous variants in TMEM161B, KIF26A, and MAN2C1, each with consistent genotype-phenotype relationships in multiple families. Conclusions and Relevance: This study's findings reveal a higher than previously recognized rate of identifiable genetic causes, specifically of channelopathies, in individuals with polymicrogyria and support the utility of exome sequencing for families affected with polymicrogyria.

Full-colour Jabuticaba-like nanostructures via the multiplex and orthogonal self-assembly of protein-conjugated quantum dots with engineered biofilms.

The integration of inorganic components with bacterial biofilms is of great significance for expanding the functionality of artificial biological materials. However, so far, the complexities and functionalities of biofilm-based scaffolds assembled via metal-peptide coordination chemistries remain limited. Here, we present a platform for the multiplexed and specific coupling of recombinant protein-functionalized fluorescent red-green-blue (RGB) quantum dots (QDs) with engineered biofilms to form Jabuticaba-like nanostructures. Full-color living Jabuticaba-like nanostructures have been achieved through the interaction of extracellular peptides that are fabricated by biofilms with the proteins that modify the surface of the RGB QDs through orthogonal SpyTag/SpyCatcher, IsopeptagN/PilinN, and IsopeptagC/PilinC pairs. We envision that living cell populations will enable the multiplexable, scalable and bottom-up assembly of versatile materials that integrate both abiotic and biotic components into multifunctional systems.

TNF in the era of immune checkpoint inhibitors: friend or foe?

Immune checkpoint inhibitors (ICIs) are effective in the treatment of patients with advanced cancer and have emerged as a pillar of standard cancer care. However, their use is complicated by adverse effects known as immune-related adverse events (irAEs), including ICI-induced inflammatory arthritis. ICI-induced inflammatory arthritis is distinguished from other irAEs by its persistence and requirement for long-term treatment. TNF inhibitors are commonly used to treat inflammatory diseases such as rheumatoid arthritis, spondyloarthropathies and inflammatory bowel disease, and have also been adopted as second-line agents to treat irAEs refractory to glucocorticoid treatment. Experiencing an irAE is associated with a better antitumour response after ICI treatment. However, whether TNF inhibition can be safely used to treat irAEs without promoting cancer progression, either by compromising ICI therapy efficacy or via another route, remains an open question. In this Review, we discuss clinical and preclinical studies that address the relationship between TNF, TNF inhibition and cancer. The bulk of the evidence suggests that at least short courses of TNF inhibitors are safe for the treatment of irAEs in patients with cancer undergoing ICI therapy. Data from preclinical studies hint that TNF inhibition might augment the antitumour effect of ICI therapy while simultaneously ameliorating irAEs.

Polymicrogyria is Associated With Pathogenic Variants in PTEN.

OBJECTIVE: Congenital structural brain malformations have been described in patients with pathogenic phosphatase and tensin homologue (PTEN) variants, but the frequency of cortical malformations in patients with PTEN variants and their impact on clinical phenotype are not well understood. Our goal was to systematically characterize brain malformations in patients with PTEN variants and assess the relevance of their brain malformations to clinical presentation. METHODS: We systematically searched a local radiology database for patients with PTEN variants who had available brain magnetic resonance imaging (MRI). The MRI scans were reviewed systematically for cortical abnormalities. We reviewed electroencephalogram (EEG) data and evaluated the electronic medical record for evidence of epilepsy and developmental delay. RESULTS: In total, we identified 22 patients with PTEN pathogenic variants for which brain MRIs were available (age range 0.4-17 years). Twelve among these 22 patients (54%) had polymicrogyria (PMG). Variants associated with PMG or atypical gyration encoded regions of the phosphatase or C2 domains of PTEN. Interestingly, epilepsy was present in only 2 of the 12 patients with PMG. We found a trend toward higher rates of global developmental delay (GDD), intellectual disability (ID), and motor delay in individuals with cortical abnormalities, although cohort size limited statistical significance. INTERPRETATION: Malformations of cortical development, PMG in particular, represent an under-recognized phenotype associated with PTEN pathogenic variants and may have an association with cognitive and motor delays. Epilepsy was infrequent compared to the previously reported high risk of epilepsy in patients with PMG. ANN NEUROL 2020;88:1153-1164.

Galangin, a Flavonoid from Lesser Galangal, Induced Apoptosis via p53-Dependent Pathway in Ovarian Cancer Cells.

Among women worldwide, ovarian cancer is one of the most dangerous cancers. Patients undergoing platinum-based chemotherapy might get adverse side effects and develop resistance to drugs. In recent years, natural compounds have aroused growing attention in cancer treatment. Galangin inhibited the growth of two cell lines, A2780/CP70 and OVCAR-3, more strongly than the growth of a normal ovarian cell line, IOSE 364. The IC50 values of galangin on proliferation of A2780/CP70, OVCAR-3 and IOSE 364 cells were 42.3, 34.5, and 131.3 µM, respectively. Flow cytometry analysis indicated that galangin preferentially induced apoptosis in both ovarian cancer cells with respect to normal ovarian cells. Galangin treatment increased the level of cleaved caspase-3 and -7 via the p53-dependent intrinsic apoptotic pathway by up-regulating Bax protein and via the p53-dependent extrinsic apoptotic pathway by up-regulating DR5 protein. By down-regulating the level of p53 with 20 µM pifithrin-α (PFT-α), the apoptotic rates of OVCAR-3 cells induced by galangin treatment (40 µM) were significantly decreased from 18.2% to 10.2%, indicating that p53 is a key regulatory protein in galangin-induced apoptosis in ovarian cancer cells. Although galangin up-regulated the expression of p21, it had little effect on the cell cycle of the two ovarian cancer cell lines. Furthermore, the levels of phosphorylated Akt and phosphorylated p70S6K were decreased through galangin treatment, suggesting that the Akt/p70S6K pathways might be involved in the apoptosis. Our results suggested that galangin is selective against cancer cells and can be used for the treatment of platinum-resistant ovarian cancers in humans.

Sodium Channel SCN3A (NaV1.3) Regulation of Human Cerebral Cortical Folding and Oral Motor Development.

Channelopathies are disorders caused by abnormal ion channel function in differentiated excitable tissues. We discovered a unique neurodevelopmental channelopathy resulting from pathogenic variants in SCN3A, a gene encoding the voltage-gated sodium channel NaV1.3. Pathogenic NaV1.3 channels showed altered biophysical properties including increased persistent current. Remarkably, affected individuals showed disrupted folding (polymicrogyria) of the perisylvian cortex of the brain but did not typically exhibit epilepsy; they presented with prominent speech and oral motor dysfunction, implicating SCN3A in prenatal development of human cortical language areas. The development of this disorder parallels SCN3A expression, which we observed to be highest early in fetal cortical development in progenitor cells of the outer subventricular zone and cortical plate neurons and decreased postnatally, when SCN1A (NaV1.1) expression increased. Disrupted cerebral cortical folding and neuronal migration were recapitulated in ferrets expressing the mutant channel, underscoring the unexpected role of SCN3A in progenitor cells and migrating neurons.

Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays.

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

Synthetic Biogenesis of Bacterial Amyloid Nanomaterials with Tunable Inorganic-Organic Interfaces and Electrical Conductivity.

Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin subunit CsgA, and forms biological nanofibers with high aspect ratios. Here, we explore the application of curli fibers for nanotechnology by engineering curli to mediate tunable biological interfaces with inorganic materials and to controllably form gold nanoparticles and gold nanowires. Specifically, we used cell-synthesized curli fibers as templates for nucleating and growing gold nanoparticles and showed that nanoparticle size could be modulated as a function of curli fiber gold-binding affinity. Furthermore, we demonstrated that gold nanoparticles can be preseeded onto curli fibers and followed by gold enhancement to form nanowires. Using these two approaches, we created artificial cellular systems that integrate inorganic-organic materials to achieve tunable electrical conductivity. We envision that cell-synthesized amyloid nanofibers will be useful for interfacing abiotic and biotic systems to create living functional materials..

The Extraction, Anticancer Effect, Bioavailability, and Nanotechnology of Baicalin.

The dried root of Baikal skullcap (Scutellaria baicalensis) has been historically and widely used in traditional Eastern medicine. Modern science proved that baicalin is the major bioactive responsible for the physiological activity of Baikal skullcap. Baicalin, a flavonoid found in several species in the genus Scutellaria, has been regarded as a potent anticancer agent. In this review, we present the main extraction methods, anticancer activity and bioavailability of baicalin. Besides, the utilization of nanotechnology to improve the bioavailability of baicalin is also mentioned.

Gallic acid, a phenolic compound, exerts anti-angiogenic effects via the PTEN/AKT/HIF-1α/VEGF signaling pathway in ovarian cancer cells.

Gallic acid (GA), a polyphenol, is widely found in numerous fruits and vegetables, particularly in hickory nuts. In the present study, we found that gallic acid, a natural phenolic compound isolated from fruits and vegetables, had a more potent growth inhibitory effect on two ovarian cancer cell lines, OVCAR-3 and A2780/CP70, than the effect on a normal ovarian cell line, IOSE-364. These results demonstrated that GA selectively inhibits the growth of cancer cells. Gene expression was examined by ELISA and western blot analysis, and gene pathways were examined by luciferase assay. It was found that GA inhibited VEGF secretion and suppressed in vitro angiogenesis in a concentration-dependent manner. GA downregulated AKT phosphorylation as well as HIF-1α expression but promoted PTEN expression. The luciferase assay results suggest that the PTEN/AKT/HIF-1α pathway accounts for the inhibitory effect of GA on VEGF expression and in vitro angiogenesis. These findings provide strong support for the high potential of GA in the prevention and therapy of ovarian cancer.

Myricetin inhibits proliferation of cisplatin-resistant cancer cells through a p53-dependent apoptotic pathway.

Cisplatin is a commonly used drug for cancer treatment by crosslinking DNA, leading to apoptosis of cancer cells, resistance to cisplatin treatment often occurs, leading to relapse. Therefore, there is a need for the development of more effective treatment strategies that can overcome chemoresistance. Myricetin is a flavonoid from fruits and vegetables, showing anticancer activity in various cancer cells. In this study, we found myricetin exhibited greater cytotoxicity than cisplatin in two cisplatin-resistant ovarian cancer cell lines, OVCAR-3 and A2780/CP70, and it was less cytotoxic to the normal ovarian cell line IOSE-364. Myricetin selectively induced apoptosis in both cisplatin-resistant cancer cell lines, but did not induce apoptosis in the normal ovarian cell line. It induced both Bcl-2 family-dependent intrinsic and DR5 dependent extrinsic apoptosis in OVCAR-3 cells. P53, a multifunctional tumor suppressor, regulated apoptosis in OVCAR-3 cells through a Bcl-2 family protein-dependent pathway. Myricetin did not induce cell cycle arrest in either ovarian cancer cell line. Because of its potency and selectivity against cisplatin-resistant cancer cells, myricetin could potentially be used to overcome cancer chemoresistance against platinum-based therapy.

Dietary compounds galangin and myricetin suppress ovarian cancer cell angiogenesis.

Galangin and myricetin are flavonoids isolated from vegetables and fruits which exhibit anti-proliferative activity in human cancer cells. In this study, their anti-angiogenic effects were investigated with in vitro (HUVEC) and in vivo (CAM) models, which showed that galangin and myricetin inhibited angiogenesis induced by OVCAR-3 cells. The molecular mechanisms through which galangin and myricetin suppress angiogenesis were also studied. It was observed that galangin and myricetin inhibited secretion of the key angiogenesis mediator vascular endothelial growth factor (VEGF) and decreased levels of p-Akt, p-70S6K and hypoxia-inducible factor-1α (HIF-1α) proteins in A2780/CP70 and OVCAR-3 cells. Transient transfection experiments showed that galangin and myricetin inhibited secretion of VEGF by the Akt/p70S6K/ HIF-1α pathway. Moreover, a novel pathway, p21/HIF-1α/VEGF, was found to be involved in the inhibitory effect of myricetin on angiogenesis in OVCAR-3 cells. These data suggest that galangin and myricetin might serve as potential anti-angiogenic agents in the prevention of ovarian cancers dependent on new blood vessel networks.

The flavonoid nobiletin inhibits tumor growth and angiogenesis of ovarian cancers via the Akt pathway.

Despite its importance, the death rate of ovarian cancer has remained unchanged over the past five decades, demanding an improvement in prevention and treatment of this malignancy. With no known carcinogens, targeted prevention is currently unavailable, and efforts in early detection of this malignancy by screening biomarkers have failed. The inhibition of angiogenesis, also known as angioprevention, is a promising strategy to limit the growth of solid tumors, including ovarian cancers. Nobiletin, a polymethoxy flavonoid compound isolated from the tiansheng plant, has been shown to inhibit the growth of multiple types of human cancers. However, there are no reports involving the effect on nobiletin on human ovarian cancer. The present report shows that nobiletin potently decreases the viability of ovarian cancer cells in vitro. However, nobiletin does not affect the viability of normal ovarian epithelial cells at <40 µM. The antitumor activity of nobiletin was also observed in athymic mouse models and in chicken chorioallantoic membrane (CAM) models. The anti-neoplastic activity of nobiletin was due to its ability to inhibit angiogenesis. We also studied the molecular mechanisms by which nobiletin suppresses angiogenesis. We observed that nobiletin inhibits secretion of the key angiogenesis mediators, Akt, HIF-1α, NF-κB and vascular epithelial growth factor (VEGF) by ovarian cancer cells. Transient transfection experiments showed that nobiletin inhibits production of HIF-1α by downregulation of Akt. Such decreased levels of HIF-1α were responsible for nobiletin-induced suppression of VEGF. Our data suggest that nobiletin may be a promising anti-angiogenic agent relevant for therapy of ovarian cancers.

Engineering living functional materials.

Natural materials, such as bone, integrate living cells composed of organic molecules together with inorganic components. This enables combinations of functionalities, such as mechanical strength and the ability to regenerate and remodel, which are not present in existing synthetic materials. Taking a cue from nature, we propose that engineered 'living functional materials' and 'living materials synthesis platforms' that incorporate both living systems and inorganic components could transform the performance and the manufacturing of materials. As a proof-of-concept, we recently demonstrated that synthetic gene circuits in Escherichia coli enabled biofilms to be both a functional material in its own right and a materials-synthesis platform. To demonstrate the former, we engineered E. coli biofilms into a chemical-inducer-responsive electrical switch. To demonstrate the latter, we engineered E. coli biofilms to dynamically organize biotic-abiotic materials across multiple length scales, template gold nanorods, gold nanowires, and metal/semiconductor heterostructures, and synthesize semiconductor nanoparticles (Chen, A. Y. et al. (2014) Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 13, 515-523.). Thus, tools from synthetic biology, such as those for artificial gene regulation, can be used to engineer the spatiotemporal characteristics of living systems and to interface living systems with inorganic materials. Such hybrids can possess novel properties enabled by living cells while retaining desirable functionalities of inorganic systems. These systems, as living functional materials and as living materials foundries, would provide a radically different paradigm of materials performance and synthesis-materials possessing multifunctional, self-healing, adaptable, and evolvable properties that are created and organized in a distributed, bottom-up, autonomously assembled, and environmentally sustainable manner.

Nobiletin suppresses cell viability through AKT pathways in PC-3 and DU-145 prostate cancer cells.

BACKGROUND: Nobiletin is a non-toxic dietary flavonoid that possesses anti-cancer properties. Nobiletin has been reported to reduce the risk of prostate cancer, but the mechanism is not well understood. In this study, we investigated the effects of nobiletin in prostate cancer cell lines PC-3 and DU-145. METHODS: Nobiletin was isolated from a polymethoxy flavonoid mixture using HPLC, cell viability was analyzed with MTS-based assays. Protein expression was examined by ELISA and western blotting. Gene expression was examined by luciferase assay. And the pathways were examined by manipulating genetic components with plasmid transfection. RESULTS: Data showed that nobiletin decreased cell viability in both prostate cell lines, with a greater reduction in viability in PC-3 cells. HIF-1α expression and AKT phosphorylation were decreased in both cell lines. The VEGF expression was inhibited in PC-3 but not DU-145 cells. cMyc expression was decreased in DU-145 cells. Nobiletin down-regulated NF-κB (p50) expression in nuclei of DU145 cells but not whole cells. It also suppressed NF-κB expression in both whole cells and nuclei of PC-3 cells. Increasing HIF-1α levels reversed nobiletin's inhibitory effects on VEGF expression, and up-regulating AKT levels reversed its inhibitory effects on HIF-1α expression. We speculate that AKT influences cell viability probably by its effect on NF-κB in both prostate cells. The effect of nobiletin on VEGF expression in PC-3 cell lines was through the AKT/HIF-1α pathway. CONCLUSION: Taken together, our results show that nobiletin suppresses cell viability through AKT pathways, with a more profound effect against the more metastatic PC-3 line. Due to this enhanced action against a more malignant cell type, nobiletin may be used to improve prostate cancer survival rates.

Synthesis and patterning of tunable multiscale materials with engineered cells.

Many natural biological systems--such as biofilms, shells and skeletal tissues--are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.

A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention.

Kaempferol is a polyphenol antioxidant found in fruits and vegetables. Many studies have described the beneficial effects of dietary kaempferol in reducing the risk of chronic diseases, especially cancer. Epidemiological studies have shown an inverse relationship between kaempferol intake and cancer. Kaempferol may help by augmenting the body's antioxidant defence against free radicals, which promote the development of cancer. At the molecular level, kaempferol has been reported to modulate a number of key elements in cellular signal transduction pathways linked to apoptosis, angiogenesis, inflammation, and metastasis. Significantly, kaempferol inhibits cancer cell growth and angiogenesis and induces cancer cell apoptosis, but on the other hand, kaempferol appears to preserve normal cell viability, in some cases exerting a protective effect. The aim of this review is to synthesize information concerning the extraction of kaempferol, as well as to provide insights into the molecular basis of its potential chemo-preventative activities, with an emphasis on its ability to control intracellular signaling cascades that regulate the aforementioned processes. Chemoprevention using nanotechnology to improve the bioavailability of kaempferol is also discussed.

Inhibitory effect of baicalin and baicalein on ovarian cancer cells.

Ovarian cancer is one of the primary causes of death for women all through the Western world. Baicalin and baicalein are naturally occurring flavonoids that are found in the roots and leaves of some Chinese medicinal plants and are thought to have antioxidant activity and possible anti-angiogenic, anti-cancer, anxiolytic, anti-inflammatory and neuroprotective activities. Two kinds of ovarian cancer (OVCAR-3 and CP-70) cell lines and a normal ovarian cell line (IOSE-364) were selected to be investigated in the inhibitory effect of baicalin and baicalein on cancer cells. Largely, baicalin and baicalein inhibited ovarian cancer cell viability in both ovarian cancer cell lines with LD50 values in the range of 45-55 µM for baicalin and 25-40 µM for baicalein. On the other hand, both compounds had fewer inhibitory effects on normal ovarian cells viability with LD50 values of 177 µM for baicalin and 68 µM for baicalein. Baicalin decreased expression of VEGF (20 µM), cMyc (80 µM), and NFkB (20 µM); baicalein decreased expression of VEGF (10 µM), HIF-1α (20 µM), cMyc (20 µM), and NFkB (40 µM). Therefore baicalein is more effective in inhibiting cancer cell viability and expression of VEGF, HIF-1α, cMyc, and NFκB in both ovarian cancer cell lines. It seems that baicalein inhibited cancer cell viability through the inhibition of cancer promoting genes expression including VEGF, HIF-1α, cMyc, and NFκB. Overall, this study showed that baicalein and baicalin significantly inhibited the viability of ovarian cancer cells, while generally exerting less of an effect on normal cells. They have potential for chemoprevention and treatment of ovarian cancers.