Implanted synthetic cells trigger tissue angiogenesis through de novo production of recombinant growth factors.

Progress in bottom-up synthetic biology has stimulated the development of synthetic cells (SCs), autonomous protein-manufacturing particles, as dynamic biomimetics for replacing diseased natural cells and addressing medical needs. Here, we report that SCs genetically encoded to produce proangiogenic factors triggered the physiological process of neovascularization in mice. The SCs were constructed of giant lipid vesicles and were optimized to facilitate enhanced protein production. When introduced with the appropriate genetic code, the SCs synthesized a recombinant human basic fibroblast growth factor (bFGF), reaching expression levels of up to 9⋅106 protein copies per SC. In culture, the SCs induced endothelial cell proliferation, migration, tube formation, and angiogenesis-related intracellular signaling, confirming their proangiogenic activity. Integrating the SCs with bioengineered constructs bearing endothelial cells promoted the remodeling of mature vascular networks, supported by a collagen-IV basement membrane-like matrix. In vivo, prolonged local administration of the SCs in mice triggered the infiltration of blood vessels into implanted Matrigel plugs without recorded systemic immunogenicity. These findings emphasize the potential of SCs as therapeutic platforms for activating physiological processes by autonomously producing biological drugs inside the body.

Bullying perpetration and victimization associations to suicide behavior: a longitudinal study.

Previous studies have reported a longitudinal association between cybervictimization and suicidal thoughts and behavior. However, the relationship between cyber-perpetration and prospective suicide risk remains unclear. The sample was composed of 2150 at-risk adolescents (mean age 15.42), enrolled in Vocational Education and Training high schools in Israel. Cyberbullying, traditional bullying, depression, hostility, serious suicidal ideations, and suicide attempts were assessed through self-report questionnaires at the beginning of the school year and one year later. All types of victimization and preparation were cross-sectionally associated with suicide ideation and attempts. Longitudinal associations were found between cyber-perpetration and suicidal ideation/attempts. Cyber-perpetrators were found to be over twice more likely to report serious suicidal ideation (OR = 2.04) or attempt suicide (OR = 2.64) in the subsequent year compared to noninvolved adolescents. These associations were significant even after adjusting for baseline depression, hostility, and traditional bullying. Traditional bullying perpetration was prospectively associated with suicide attempts. Traditional victimization was cross-sectionally associated with suicide ideation and attempts but not prospectively. Cybervictimization was prospectively associated with suicide ideation but not to suicide attempts. The findings demonstrate the prospective risk of involvement in bullying in regard to suicide ideation and behavior. Cyberbullying was found to be a somewhat differentiated phenomena from traditional bullying.

Time multiplexing super-resolved imaging without a priori knowledge of the spatial distribution of the encoding structured illumination.

Time multiplexing is a super-resolution technique that sacrifices time to overcome the resolution reduction obtained because of diffraction. There are many super resolution methods based on time multiplexing, but all of them require a priori knowledge of the time changing encoding mask, which is projected on the object and used to encode and decode the high-resolution information. In this paper, we present a time multiplexing technique that does not require the a priori knowledge on the projected encoding mask. First, the theoretical concept of the technique is demonstrated; then, numerical simulations and experimental results are presented.

Superresolved space-dependent sensing of temporal signals by space multiplexing.

In this paper we present a novel, to the best of our knowledge, superresolved sensing concept aiming to improve the resolution of temporal signals obtained by applying space multiplexing. The system's temporal resolution is limited due to the sensor's time averaging. The proposed superresolution technique overcomes this time average and can therefore increase the system's temporal resolution. In addition, we also show how to increase the temporal sampling rate, which also affects the temporal resolution. The proposed concept requires that the object will be fixed in space in order to apply the space multiplexing. It can especially be relevant for superresolving laser-based time-of-flight sensors and images as well as light detection and ranging (lidar) devices.

Chemical Zymogens and Transmembrane Activation of Transcription in Synthetic Cells.

In this work, synthetic cells equipped with an artificial signaling pathway that connects an extracellular trigger event to the activation of intracellular transcription are engineered. Learning from nature, this is done via an engineering of responsive enzymes, such that activation of enzymatic activity can be triggered by an external biochemical stimulus. Reversibly deactivated creatine kinase to achieve triggered production of adenosine triphosphate, and a reversibly deactivated nucleic acid polymerase for on-demand synthesis of RNA are engineered. An extracellular, enzyme-activated production of a diffusible zymogen activator is also designed. The key achievement of this work is that the importance of cellularity is illustrated whereby the separation of biochemical partners is essential to resolve their incompatibility, to enable transcription within the confines of a synthetic cell. The herein designed biochemical pathway and the engineered synthetic cells are arguably primitive compared to their natural counterpart. Nevertheless, the results present a significant step toward the design of synthetic cells with responsive behavior, en route from abiotic to life-like cell mimics.

Polymersomes with splenic avidity target red pulp myeloid cells for cancer immunotherapy.

Regulating innate immunity is an emerging approach to improve cancer immunotherapy. Such regulation requires engaging myeloid cells by delivering immunomodulatory compounds to hematopoietic organs, including the spleen. Here we present a polymersome-based nanocarrier with splenic avidity and propensity for red pulp myeloid cell uptake. We characterized the in vivo behaviour of four chemically identical yet topologically different polymersomes by in vivo positron emission tomography imaging and innovative flow and mass cytometry techniques. Upon intravenous administration, relatively large and spherical polymersomes accumulated rapidly in the spleen and efficiently targeted myeloid cells in the splenic red pulp. When loaded with ß-glucan, intravenously administered polymersomes significantly reduced tumour growth in a mouse melanoma model. We initiated our nanotherapeutic's clinical translation with a biodistribution study in non-human primates, which revealed that the platform's splenic avidity is preserved across species.

Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

Synthetic cells with self-activating optogenetic proteins communicate with natural cells.

Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells' capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA→RNA→protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

Integrating Artificial Intelligence and Nanotechnology for Precision Cancer Medicine.

Artificial intelligence (AI) and nanotechnology are two fields that are instrumental in realizing the goal of precision medicine-tailoring the best treatment for each cancer patient. Recent conversion between these two fields is enabling better patient data acquisition and improved design of nanomaterials for precision cancer medicine. Diagnostic nanomaterials are used to assemble a patient-specific disease profile, which is then leveraged, through a set of therapeutic nanotechnologies, to improve the treatment outcome. However, high intratumor and interpatient heterogeneities make the rational design of diagnostic and therapeutic platforms, and analysis of their output, extremely difficult. Integration of AI approaches can bridge this gap, using pattern analysis and classification algorithms for improved diagnostic and therapeutic accuracy. Nanomedicine design also benefits from the application of AI, by optimizing material properties according to predicted interactions with the target drug, biological fluids, immune system, vasculature, and cell membranes, all affecting therapeutic efficacy. Here, fundamental concepts in AI are described and the contributions and promise of nanotechnology coupled with AI to the future of precision cancer medicine are reviewed.

Wireless Optogenetic Stimulation of Oxytocin Neurons in a Semi-natural Setup Dynamically Elevates Both Pro-social and Agonistic Behaviors.

Complex behavioral phenotyping techniques are becoming more prevalent in the field of behavioral neuroscience, and thus methods for manipulating neuronal activity must be adapted to fit into such paradigms. Here, we present a head-mounted, magnetically activated device for wireless optogenetic manipulation that is compact, simple to construct, and suitable for use in group-living mice in an enriched semi-natural arena over several days. Using this device, we demonstrate that repeated activation of oxytocin neurons in male mice can have different effects on pro-social and agonistic behaviors, depending on the social context. Our findings support the social salience hypothesis of oxytocin and emphasize the importance of the environment in the study of social neuromodulators. Our wireless optogenetic device can be easily adapted for use in a variety of behavioral paradigms, which are normally hindered by tethered light delivery or a limited environment.

The Association Between Gender, Ethnicity, and Suicidality Among Vocational Students in Israel.

Gender and ethnicity are significant factors when evaluating suicidal risk, especially among ethnically diverse populations. In the current study we explored the association between gender, ethnicity, and suicide ideation and attempts among Arab and Jewish vocational education and training high school students in Israel. Students (N = 3,554) completed a self-report survey evaluating suicide ideation and attempts, depression, anxiety, somatization, and sense of belonging. Hierarchical generalized linear modeling indicated that female Arab adolescents had elevated levels of suicide ideation, higher rates of suicide attempts, and greater psychological distress than Arab males and Jewish students. Furthermore, female Arab adolescents were found to be more susceptible to suicide ideation when depression levels were high. These results are discussed in the context of the double-jeopardy Arab young women face, as members of a minority ethnic group in Israel and their status as women within the patriarchal Arab culture.

Religious-Ethnic Differences in Prevalence and Correlates of Suicidal Ideation and Attempts Among Israeli Vocational Education Students.

BACKGROUND: The current study is the first to examine serious suicidal ideation, suicide attempts and psychological distress among students belonging to different religious-ethnic groups attending VET high schools. METHOD: 3,553 students studying in the VET high schools completed a self-report survey. The survey evaluated serious suicidal ideation, suicide attempts, depression, anxiety, somatization, self-efficacy, belonging and sociodemographic variables. RESULTS: Students coming from minority groups (Arab Muslim, Bedouin Arab Muslim, Druze) are more likely to have seriously considered suicide or attempted suicide compared to their Jewish counterparts, with the exception of Christian Arabs. Bedouin Arab Muslim students were more susceptible to suicide ideation and attempts upon high depression levels. CONCLUSIONS: Minority youth at VET schools are at greater risk of experiencing suicidal ideation and attempting suicide compared to non-minority students. The education system must be highly alert to depressive symptoms among Bedouin Arab Muslim students since they may pose a greater risk to develop into suicidal ideation and attempt.

Ovarian angiotensin-converting enzyme activity in humans: relationship to estradiol, age, and uterine pathology.

The present study was designed to measure angiotensin-converting enzyme (ACE) activity in the human ovary and in serum and to relate this activity to age, serum estradiol levels, and uterine and endometrial pathology. ACE activity was determined in 56 females by a radiometric assay using [3H]hippuryl-glycyl-glycine as substrate. Ovarian ACE activity, but not serum ACE, was found to increase with age (P < 0.01) and was significantly greater in postmenopausal subjects (n = 31; 1.35 +/- 0.05 nmol/mg.min) than in subjects with active ovaries (n = 21; 0.65 +/- 0.2 nmol/mg.min; P = 0.0033). Ovarian ACE activities in fertile women in the preovulatory phase (n = 14) and the postovulatory phase (n = 7) were not statistically different (0.66 +/- 0.23 and 0.63 +/- 0.17 nmol/mg.min, respectively). Serum ACE activities were similar in females with active and nonactive ovaries (87.6 +/- 5.0 vs. 81.7 +/- 5.3 nmol/mL-min, respectively). Serum estradiol levels in fertile women were significantly higher than those in postmenopausal women (P = 0.0023). Serum estradiol levels were negatively correlated with age (r = -0.46; P = 0.0041) and were not correlated with either serum ACE activity (r = 0.080; P = NS) or ovarian ACE activity. In summary, human ovarian ACE activity, but not serum ACE, is positively correlated with age. Serum estradiol levels decrease with age, but are not correlated with either ovarian or serum ACE activity. Endogenous serum estradiol levels had no apparent effect on ovarian or serum ACE activity. The presence of uterine pathology affects ovarian ACE activity. The cause of the increased ovarian ACE activity is not clear, but may be related to the aging process.

Inhibition of angiotensin converting enzyme by ramipril in serum and tissue of man.

Studies in animal models have indicated that ramipril is a potent inhibitor of angiotensin converting enzyme (ACE) in serum and tissue. In our study, the normal range of ACE activity and the inhibitory effect of short-term oral administration of ramipril on ACE activity in human serum and tissue samples of renal cortex, heart and blood vessels were determined. ACE activity in the renal cortex (125.2 +/- 11.5 nmol/mg per min) was greater than 600 times that of the heart (0.20 +/- 0.01 nmol/mg per min), greater than 500 times that of the veins (0.23 +/- 0.09 nmol/mg per min) and greater than 150 times that of the arteries (0.80 +/- 0.23 nmol/mg per min). ACE activity in the renal cortex and arteries 2 h after last dosing was almost completely inhibited by ramipril whereas ACE activity in the veins and heart was inhibited to a lesser extent. Our results demonstrate in man, for the first time, an inhibition of tissue ACE following short-term oral treatment with an ACE inhibitor.

The role of eicosanoids in cyclosporine nephrotoxicity in the rat.

Nephrotoxicity is the most troublesome complication of cyclosporine (CSA) therapy. The present study was designed to investigate the effects of chronic treatment with CSA on the 24-hr urinary excretion of prostanoids (PGs) and thromboxane (Tx) and on the renal function in the absence or presence of indomethacin. CSA administration to Wistar rats (20 mg/kg/day, i.p.) for 14 days caused a significant increase in plasma creatinine, blood urea nitrogen (BUN), urine osmolality, fractional excretion of sodium and potassium and a reduction in creatinine clearance (CCr) and urine volume. These changes were associated with a significant reduction in urinary excretion of PGE2 (21.1 +/- 3.3 vs 33.0 +/- 2.5 ng/24 hr) and PGF2 alpha (13.4 +/- 1.4 vs 27.9 +/- 3.8 ng/24 hr) and an increase in TxB2 (12.1 +/- 3.0 vs 4.6 +/- 0.5 ng/24 hr), and 6-keto PGF1 alpha (56.2 +/- 7.7 vs 27.7 +/- 1.9 ng/24 hr). However, the synthesis of TxB2 and 6-keto PGF1 alpha by renal medullary and cortical slices prepared from CSA treated rats was not different from values obtained for vehicle treatment. In contrast, PGE2 synthesis by cortical slices prepared from the CSA group was increased. A single injection of indomethacin (10 mg/kg) to vehicle and CSA treated rats resulted in a significant reduction in PGs and TxB2 excretion. This, was associated with a further reduction in CCr (0.81 +/- 0.06 vs 1.03 +/- 0.04 ml/min) and an increase in BUN (38.5 +/- 5.2 vs 28.2 +/- 1.4 mg%) only in the CSA group. We suggest that the vasodilating PGs attenuate the renal toxic effects induced by CSA.

Cyclosporin treatment alters prostanoid and thromboxane production by rat isolated kidney mitochondria.

This study was designed to investigate the effects of chronic treatment with cyclosporin A (CSA) on the endogenous synthesis of prostanoids (PGs) and thromboxane (Tx) by renal isolated medullary and cortical mitochondria. The administration of CSA, dissolved in 10% ethanol in olive oil, to male Wistar rats (20 mg kg-1 day-1 i.p.) for 14 days resulted in alterations in mitochondrial biosynthesis of immunoreactive PGs. The endogenous synthesis of thromboxane by medullary and cortical mitochondria isolated from CSA-treated rats was significantly enhanced by 120 and 55%, respectively, whereas the synthesis of prostaglandin E2 by medullary mitochondria was reduced by 35%. The synthesis of prostaglandin F2 alpha and prostacyclin was not affected by CSA treatment. The conversion of exogenous arachidonic acid to PGs and Tx by cortical mitochondria isolated from CSA-treated rats was significantly increased. In addition, CSA treatment resulted in i) a reduced acylation of arachidonic acid into medullary phospholipids by 25% and into medullary and cortical triglycerides by 33 and 27%, respectively, and ii) an increase in cortical and medullary triglycerides. We suggest that the alterations in the endogenous mitochondrial production of PGs and Tx caused by CSA, may play a role in the impairment of membrane mediated functions.

Cyclosporin A treatment enhances angiotensin converting enzyme activity in lung and serum of rats.

Nephrotoxicity and arterial hypertension are the most common side effects of treatment with cyclosporin A (CSA). Its effects on angiotensin converting enzyme (ACE) activity in the renal cortex, lung and serum of nephrotoxic rats have been investigated. Wistar rats were treated with CSA (20 mg kg-1 day-1 i.p.) or vehicle (olive oil containing 10% ethanol) for 14 days. On day 15, the rats were killed and ACE activity determined by radiometric assay using [3H]hippuryl-glycyl-glycine as substrate. CSA treatment resulted in a decrease in creatinine clearance, urine flow and body weight and a significant increase in serum and lung ACE activities (436 +/- 9 vs 391 +/- 7 nmol mL-1 min-1, P less than 0.001; 184 +/- 8 vs 142 +/- 10 nmol mg-1 min-1 P less than 0.01, respectively). In contrast, renal cortex ACE activity was reduced in the CSA-treated rats (0.35 +/- 0.02 vs 0.51 +/- 0.02 nmol mg-1 min-1, P less than 0.01). ACE activities in the renal cortex and serum were not affected by treatment with gentamicin (80 mg kg-1 day-1) for 11 days. In rats treated simultaneously with CSA and captopril (50 mg kg-1 day-1) ACE activity in the serum, lung and renal cortex was inhibited by 95, 93 and 92%, respectively. These changes in ACE activity were associated with a decreased systolic blood pressure in the rats receiving CSA and captopril. Therefore, ACE activity in the serum and lung of CSA-treated rats was increased, while its activity in the renal cortex was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostanoids and thromboxane production by renal brush border membrane.

It has been estimated that proximal tubule are responsible for about 7.5% of total kidney prostaglandin PGE2 production. In the present report we investigated the production of prostanoids and thromboxane by rabbit renal proximal tubule brush border membrane. PGF2 alpha was the major endogenous prostaglandin produced under basal condition. The addition of exogenous arachidonic acid increased only PGE2 production. No PGE2 production was found when vesicles were incubated with indomethacin or at 4 degrees C, suggesting the involvement of the PGH2 synthase and PGE2 isomerase enzymes. Addition of angiotensin II at 10(-6) and 10(-9) mol l-1 did not affect the endogenous PGE2 production by brush border membrane. Thus, results of our study demonstrates that the renal proximal tubule brush border membrane has the capacity to produce prostanoids and thromboxane.

Angiotensin converting enzyme activity in the serum, lung and kidney of diabetic rats.

Diabetes Mellitus in its early stages, is associated with kidney enlargement and increased glomerular filtration rate in humans and in rats. The present study was designed to clarify the direct effect of diabetes on serum and tissue angiotensin converting enzyme (ACE) activity in streptozotocin-induced diabetic rats. Serum ACE activity, as determined using a radiometric assay, was significantly increased in the diabetic rats (n = 15) 14 days after induction of diabetes (670 +/- 31 vs. 506 +/- 14 nmol ml-1 min-1). Lung ACE activity, but not renal, was significantly elevated at 7 and 14 days by 29 and 46%, respectively. Plasma renin activity in the diabetic rats was decreased at 7 and 14 days by 41 and 78%, respectively. Incubations of lung slices in the presence of glucose at different concentrations did not affect in-vitro release of the enzyme. Administration of insulin (8 units kg-1) to diabetic rats (n = 6) on the 4th day for 11 days reduced ACE activity to values below control. Thus, serum and lung ACE activity is increased in the diabetic rat and reduced upon insulin treatment.