Mapping the slow and fast photoresponse of field-effect transistors to terahertz and infrared radiation.

Field-effect transistors are capable of detecting electromagnetic radiation from less than 100 GHz up to very high frequencies reaching well into the infrared spectral range. Here, we report on frequency coverage of up to 30THz, thus reaching the technologically important frequency regime of CO2 lasers, using GaAs/AlGaAs high-electron-mobility transistors. A detailed study of the speed and polarization dependence of the responsivity allows us to identify a cross over of the dominant detection mechanism from ultrafast non-quasistatic rectification at low Terahertz frequencies to slow rectification based on a combination of the Seebeck and bolometric effects at high frequencies, occurring at about the boundary between the Terahertz frequency range and the infrared at 10THz.

Toxicologic Pathology Forum: Opinion on Interpretive Challenges for Procedure-Related Effects Associated With Direct Central Nervous System Delivery of Oligonucleotides to Rodents, Dogs, and Nonhuman Primates.

Direct delivery of therapeutics to the central nervous system (CNS) greatly expands opportunities to treat neurological diseases but is technically challenging. This opinion outlines principal technical aspects of direct CNS delivery via intracerebroventricular (ICV) or intrathecal (IT) injection to common nonclinical test species (rodents, dogs, and nonhuman primates) and describes procedure-related clinical and histopathological effects that confound interpretation of test article-related effects. Direct dosing is by ICV injection in mice due to their small body size, while other species are dosed IT in the lumbar cistern. The most frequent procedure-related functional effects are transient absence of lower spinal reflexes after IT injection or death soon after ICV dosing. Common procedure-related microscopic findings in all species include leukocyte infiltrates in CNS meninges or perivascular (Virchow-Robin) spaces; nerve fiber degeneration in the spinal cord white matter (especially dorsal and lateral tracts compressed by dosing needles or indwelling catheters), spinal nerve roots, and sciatic nerve; meningeal fibrosis at or near IT injection sites; hemorrhage; and gliosis. Findings typically are minimal to occasionally mild. Findings tend to be more severe and/or have a higher incidence in the spinal cord segments and spinal nerve roots at or close to the site of administration.

State-of-the-Art Room Temperature Operable Zero-Bias Schottky Diode-Based Terahertz Detector Up to 5.56 THz.

We present the characterization of a Zero-bias Schottky diode-based Terahertz (THz) detector up to 5.56 THz. The detector was operated with both a table-top system until 1.2 THz and at a Free-Electron Laser (FEL) facility at singular frequencies from 1.9 to 5.56 THz. We used two measurement techniques in order to discriminate the sub-ns-scale (via a 20 GHz oscilloscope) and the ms-scale (using the lock-in technique) responsivity. While the lock-in measurements basically contain all rectification effects, the sub-ns-scale detection with the oscilloscope is not sensitive to slow bolometric effects caused by changes of the IV characteristic due to temperature. The noise equivalent power (NEP) is 10 pW/Hz in the frequency range from 0.2 to 0.6 THz and 17 pW/Hz at 1.2 THz and increases to 0.9 µW/Hz at 5.56 THz, which is at the state of the art for room temperature zero-bias Schottky diode-based THz detectors with non-resonant antennas. The voltage and current responsivity of ∼500 kV/W and ∼100 mA/W, respectively, is demonstrated over a frequency range of 0.2 to 1.2 THz with the table-top system.

Range of Neurological Signs in Cynomolgus Monkeys After Intrathecal Bolus Administration of Antisense Oligonucleotides.

Intrathecal (IT) dosing (ie, injection into the subarachnoidal space at the lumbar region) is a common route of administration in cynomolgus monkey preclinical safety studies conducted for antisense oligonucleotides (ASO) that target central nervous system diseases. Herein we report on neurological signs that have been observed in 28 IT studies conducted in 1,016 cynomolgus monkeys. Neurological signs were classified into 5 groups: (1) A nonadverse transient absence of lower spinal reflexes. This observation occurred at low incidence in nontreated animals and in those that were injected artificial cerebrospinal fluid. The incidence increased in animals that were injected an ASO. Reflexes were present again at 24 hours or 48 hours after dosing. The incidence appeared to increase with dose. (2) Test-article-related adverse muscle tremor or muscle spasticity occurring during the injection procedure or immediately thereafter. In one-third of animals this finding responded to treatment with diazepam, in two-third it required euthanasia. (3) Neurological findings occurring between 30 minutes and 4 hours after dosing were characterized by any combination of ataxia, paresis, nystagmus, urinary incontinence, or muscle tremor. Those conditions either spontaneously resolved or they slowly worsened, eventually resulting in a poor general condition. (4) Neurological findings due to spinal cord injury were characterized by rapidly progressing paralysis of hind limbs. Magnetic resonance imaging revealed a focal hyperintense lesion, indicative of spinal cord necrosis. (5) Test-article-related adverse hind limb paresis or paralysis that occurred between 2 and 18 days after dosing. Those findings were rare and resulted in a poor general condition requiring euthanasia.

Save Your Maximum Tolerated Dose: How to Diagnose Procedure-Related Spinal Cord Lesions After Lumbar Intrathecal Bolus Administration of Oligonucleotides in Cynomolgus Monkeys.

Many potential drugs for treatment of neurodegenerative diseases, particularly antisense oligonucleotides (ASOs), are administered via lumbar intrathecal injection, because these drugs do not cross the blood-brain barrier. Intrathecal injection is a well-established method in cynomolgus monkeys, a species that is used in preclinical safety assessment when other nonrodent species cannot be used. The authors completed intrathecal ASO administration in over 30 preclinical safety studies (>1000 animals and >4500 dose administrations) during which we observed 3 cases of procedure-related spinal cord necrosis (incidence <0.1%). We describe clinical symptoms, diagnostic approaches, morphological features, and prognosis of this rare injury, and compare these findings with typical drug-related findings of ASOs dosed by intrathecal injection. The low incidence of procedure-related and dose-limiting lesions confines this analysis to a small sample set. The pattern of effects is similar across all monkeys despite differences in age, body weight, and intrathecal injection site. All 3 cases presented a combination of the following findings: blood in cerebrospinal fluid at time of injection, clinical signs that increase in severity within a day of dosing, lameness of both hind limbs, reduced muscle tone, and loss of patellar, foot grip, and/or anal reflexes. In all cases, magnetic resonance imaging (MRI) showed a linear hyperintense lesion in the lumbar spinal cord. In 2 cases, this hyperintensity was associated with evidence of spinal cord edema. We conclude that a pattern of in-life and pathology findings, including noninvasive MRI assessment, is indicative of procedure-related effects.

Coherent Terahertz Detection via Ultrafast Dynamics of Hot Dirac Fermions in Graphene.

Graphene has recently been shown to exhibit ultrafast conductivity modulation due to periodic carrier heating by either terahertz (THz) waves, leading to self-induced harmonic generation, or the intensity beat note of two-color optical radiation. We exploit the latter to realize an optoelectronic photomixer for coherent, continuous-wave THz detection, based on a photoconductive antenna with multilayer CVD-grown graphene in the gap. While for biased THz emitters the dark current would pose a serious detriment for performance, we show that this is not the case for bias-free THz detection and demonstrate detection up to frequencies of at least 700 GHz at room temperature, even without optimized tuning of the doping. We account for the photocurrent and photomixing response using detailed simulations of the time-dependent carrier distribution, which also indicate significant potential for enhancement of the sensitivity, to become competitive with well-established semiconductor photomixers.

The Na+ /H+ -exchanger (NHE1) generates pH nanodomains at focal adhesions.

Many tumor cells are characterized by an increased net acid production. They extrude the excess protons mainly through the Na(+) /H(+) -exchanger NHE1. An increased NHE1 activity elevates the metastatic potential of tumor cells. Cell migration, a key step in the metastatic cascade, requires the formation and release of integrin-mediated cell-matrix contacts (focal adhesions). As NHE1 has been localized to focal adhesion sites, the present study tests the hypothesis that NHE1 generates measurable pH nanodomains right at focal adhesions. In order to ratiometrically measure pH close to the plasma membrane, we established a novel application of the total internal reflection fluorescence microscopy (TIRFM). Human melanoma cells were transfected with DsRed2-paxillin to identify focal adhesion sites. The pH-sensitive dyes BCECF and WGA-fluorescein were used to measure the submembranous cytosolic and the pericellular pH, respectively. Distinct pH nanodomains were found at focal adhesions, particularly at those located at the cell front, where NHE1 was concentrated. These sites featured a remarkably alkaline cytosolic and an acidic pericellular pH and thus a much steeper proton gradient across the plasma membrane compared to the rest of the cell. The generation of pH nanodomains could be assigned to NHE1-mediated H(+) export because such pH domains could not be detected in NHE1-deficient cells. Given that both integrin avidity and mechanisms contributing to adhesion turnover are pH-sensitive, we propose that pH nanodomains at focal adhesions, locally created and maintained by NHE1 activity especially at the cell front, modulate adhesion dynamics in migrating cells.

Reconditioning the Neurogenic Niche of Adult Non-human Primates by Antisense Oligonucleotide-Mediated Attenuation of TGFß Signaling.

Adult neurogenesis is a target for brain rejuvenation as well as regeneration in aging and disease. Numerous approaches showed efficacy to elevate neurogenesis in rodents, yet translation into therapies has not been achieved. Here, we introduce a novel human TGFß-RII (Transforming Growth Factor-Receptor Type II) specific LNA-antisense oligonucleotide ("locked nucleotide acid"-"NVP-13"), which reduces TGFß-RII expression and downstream receptor signaling in human neuronal precursor cells (ReNcell CX® cells) in vitro. After we injected cynomolgus non-human primates repeatedly i.th. with NVP-13 in a preclinical regulatory 13-week GLP-toxicity program, we could specifically downregulate TGFß-RII mRNA and protein in vivo. Subsequently, we observed a dose-dependent upregulation of the neurogenic niche activity within the hippocampus and subventricular zone: human neural progenitor cells showed significantly (up to threefold over control) enhanced differentiation and cell numbers. NVP-13 treatment modulated canonical and non-canonical TGFß pathways, such as MAPK and PI3K, as well as key transcription factors and epigenetic factors involved in stem cell maintenance, such as MEF2A and pFoxO3. The latter are also dysregulated in clinical neurodegeneration, such as amyotrophic lateral sclerosis. Here, we provide for the first time in vitro and in vivo evidence for a novel translatable approach to treat neurodegenerative disorders by modulating neurogenesis.

Direct nanoscopic observation of plasma waves in the channel of a graphene field-effect transistor.

Plasma waves play an important role in many solid-state phenomena and devices. They also become significant in electronic device structures as the operation frequencies of these devices increase. A prominent example is field-effect transistors (FETs), that witness increased attention for application as rectifying detectors and mixers of electromagnetic waves at gigahertz and terahertz frequencies, where they exhibit very good sensitivity even high above the cut-off frequency defined by the carrier transit time. Transport theory predicts that the coupling of radiation at THz frequencies into the channel of an antenna-coupled FET leads to the development of a gated plasma wave, collectively involving the charge carriers of both the two-dimensional electron gas and the gate electrode. In this paper, we present the first direct visualization of these waves. Employing graphene FETs containing a buried gate electrode, we utilize near-field THz nanoscopy at room temperature to directly probe the envelope function of the electric field amplitude on the exposed graphene sheet and the neighboring antenna regions. Mapping of the field distribution documents that wave injection is unidirectional from the source side since the oscillating electrical potentials on the gate and drain are equalized by capacitive shunting. The plasma waves, excited at 2 THz, are overdamped, and their decay time lies in the range of 25-70 fs. Despite this short decay time, the decay length is rather long, i.e., 0.3-0.5 µm, because of the rather large propagation speed of the plasma waves, which is found to lie in the range of 3.5-7 × 106 m/s, in good agreement with theory. The propagation speed depends only weakly on the gate voltage swing and is consistent with the theoretically predicted 1 4 power law.

BACH: Grand challenge on breast cancer histology images.

Breast cancer is the most common invasive cancer in women, affecting more than 10% of women worldwide. Microscopic analysis of a biopsy remains one of the most important methods to diagnose the type of breast cancer. This requires specialized analysis by pathologists, in a task that i) is highly time- and cost-consuming and ii) often leads to nonconsensual results. The relevance and potential of automatic classification algorithms using hematoxylin-eosin stained histopathological images has already been demonstrated, but the reported results are still sub-optimal for clinical use. With the goal of advancing the state-of-the-art in automatic classification, the Grand Challenge on BreAst Cancer Histology images (BACH) was organized in conjunction with the 15th International Conference on Image Analysis and Recognition (ICIAR 2018). BACH aimed at the classification and localization of clinically relevant histopathological classes in microscopy and whole-slide images from a large annotated dataset, specifically compiled and made publicly available for the challenge. Following a positive response from the scientific community, a total of 64 submissions, out of 677 registrations, effectively entered the competition. The submitted algorithms improved the state-of-the-art in automatic classification of breast cancer with microscopy images to an accuracy of 87%. Convolutional neuronal networks were the most successful methodology in the BACH challenge. Detailed analysis of the collective results allowed the identification of remaining challenges in the field and recommendations for future developments. The BACH dataset remains publicly available as to promote further improvements to the field of automatic classification in digital pathology.

Extracellular protonation modulates cell-cell interaction mechanics and tissue invasion in human melanoma cells.

Detachment of cells from the primary tumour precedes metastatic progression by facilitating cell release into the tissue. Solid tumours exhibit altered pH homeostasis with extracellular acidification. In human melanoma, the Na+/H+ exchanger NHE1 is an important modifier of the tumour nanoenvironment. Here we tested the modulation of cell-cell-adhesion by extracellular pH and NHE1. MV3 tumour spheroids embedded in a collagen matrix unravelled the efficacy of cell-cell contact loosening and 3D emigration into an environment mimicking physiological confinement. Adhesive interaction strength between individual MV3 cells was quantified using atomic force microscopy and validated by multicellular aggregation assays. Extracellular acidification from pHe7.4 to 6.4 decreases cell migration and invasion but increases single cell detachment from the spheroids. Acidification and NHE1 overexpression both reduce cell-cell adhesion strength, indicated by reduced maximum pulling forces and adhesion energies. Multicellular aggregation and spheroid formation are strongly impaired under acidification or NHE1 overexpression. We show a clear dependence of melanoma cell-cell adhesion on pHe and NHE1 as a modulator. These effects are opposite to cell-matrix interactions that are strengthened by protons extruded via NHE1. We conclude that these opposite effects of NHE1 act synergistically during the metastatic cascade.

A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform.

Recent treatments of leukemias with chimeric antigen receptor (CAR) expressing T cells underline their impressive therapeutic potential. However, once adoptively transferred into patients, there is little scope left to shut them down after elimination of tumor cells or in case adverse side effects occur. This becomes of special relevance if they are directed against commonly expressed tumor associated antigens (TAAs) such as receptors of the ErbB family. To overcome this limitation, we recently established a modular CAR platform technology termed UniCAR. UniCARs are not directed against TAAs but instead against a unique peptide epitope on engineered recombinant targeting modules (TMs), which guide them to the target. In the absence of a TM UniCAR T cells are inactive. Thus an interruption of any UniCAR activity requires an elimination of unbound TM and the TM complexed with UniCAR T cells. Elimination of the latter one requires a disassembly of the UniCAR-TM complexes. Here, we describe a first nanobody (nb)-based TM directed against EGFR. The novel TM efficiently retargets UniCAR T cells to EGFR positive tumors and mediates highly efficient target-specific and target-dependent tumor cell lysis both in vitro and in vivo. After radiolabeling of the novel TM with 64Cu and 68Ga, we analyzed its biodistribution and clearance as well as the stability of the UniCAR-TM complexes. As expected unbound TM is rapidly eliminated while the elimination of the TM complexed with UniCAR T cells is delayed. Nonetheless, we show that UniCAR-TM complexes dissociate in vitro and in vivo in a concentration-dependent manner in line with the concept of a repeated stop and go retargeting of tumor cells via the UniCAR technology.

Retargeting of T lymphocytes to PSCA- or PSMA positive prostate cancer cells using the novel modular chimeric antigen receptor platform technology "UniCAR".

New treatment options especially of solid tumors including for metastasized prostate cancer (PCa) are urgently needed. Recent treatments of leukemias with chimeric antigen receptors (CARs) underline their impressive therapeutic potential. However CARs currently applied in the clinics cannot be repeatedly turned on and off potentially leading to severe life threatening side effects. To overcome these problems, we recently described a modular CAR technology termed UniCAR: UniCAR T cells are inert but can be turned on by application of one or multiple target modules (TMs). Here we present preclinical data summarizing the retargeting of UniCAR T cells to PCa cells using TMs directed to prostate stem cell- (PSCA) or/and prostate specific membrane antigen (PSMA). In the presence of the respective TM(s), we see a highly efficient target-specific and target-dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and PCa cell lysis both in vitro and experimental mice.

Draft Genome Sequences of Itaconate-Producing Ustilaginaceae.

Some smut fungi of the family Ustilaginaceae produce itaconate from glucose. De novo genome sequencing of nine itaconate-producing Ustilaginaceae revealed genome sizes between 19 and 25 Mbp. Comparison to the itaconate cluster of U. maydis MB215 revealed all essential genes for itaconate production contributing to metabolic engineering for improving itaconate production.

Roles of ion transport in control of cell motility.

Cell motility is an essential feature of life. It is essential for reproduction, propagation, embryonic development, and healing processes such as wound closure and a successful immune defense. If out of control, cell motility can become life-threatening as, for example, in metastasis or autoimmune diseases. Regardless of whether ciliary/flagellar or amoeboid movement, controlled motility always requires a concerted action of ion channels and transporters, cytoskeletal elements, and signaling cascades. Ion transport across the plasma membrane contributes to cell motility by affecting the membrane potential and voltage-sensitive ion channels, by inducing local volume changes with the help of aquaporins and by modulating cytosolic Ca(2+) and H(+) concentrations. Voltage-sensitive ion channels serve as voltage detectors in electric fields thus enabling galvanotaxis; local swelling facilitates the outgrowth of protrusions at the leading edge while local shrinkage accompanies the retraction of the cell rear; the cytosolic Ca(2+) concentration exerts its main effect on cytoskeletal dynamics via motor proteins such as myosin or dynein; and both, the intracellular and the extracellular H(+) concentration modulate cell migration and adhesion by tuning the activity of enzymes and signaling molecules in the cytosol as well as the activation state of adhesion molecules at the cell surface. In addition to the actual process of ion transport, both, channels and transporters contribute to cell migration by being part of focal adhesion complexes and/or physically interacting with components of the cytoskeleton. The present article provides an overview of how the numerous ion-transport mechanisms contribute to the various modes of cell motility.

Dynamic tension spectroscopy and strength of biomembranes.

Rupturing fluid membrane vesicles with a steady ramp of micropipette suction produces a distribution of breakage tensions governed by the kinetic process of membrane failure. When plotted as a function of log(tension loading rate), the locations of distribution peaks define a dynamic tension spectrum with distinct regimes that reflect passage of prominent energy barriers along the kinetic pathway. Using tests on five types of giant phosphatidylcholine lipid vesicles over loading rates(tension/time) from 0.01-100 mN/m/s, we show that the kinetic process of membrane breakage can be modeled by a causal sequence of two thermally-activated transitions. At fast loading rates, a steep linear regime appears in each spectrum which implies that membrane failure starts with nucleation of a rare precursor defect. The slope and projected intercept of this regime are set by defect size and frequency of spontaneous formation, respectively. But at slow loading rates, each spectrum crosses over to a shallow-curved regime where rupture tension changes weakly with rate. This regime is predicted by the classical cavitation theory for opening an unstable hole in a two-dimensional film within the lifetime of the defect state. Under slow loading, membrane edge energy and the frequency scale for thermal fluctuations in hole size are the principal factors that govern the level of tension at failure. To critically test the model and obtain the parameters governing the rates of transition under stress, distributions of rupture tension were computed and matched to the measured histograms through solution of the kinetic master (Markov) equations for defect formation and annihilation or evolution to an unstable hole under a ramp of tension. As key predictors of membrane strength, the results for spontaneous frequencies of defect formation and hole edge energies were found to correlate with membrane thicknesses and elastic bending moduli, respectively.