Implantable neural probes are widely used to record and stimulate neural activities. These probes should be stiff enough for insertion. However, it should also be flexible to minimize tissue damage after insertion. Therefore, having dynamic control of the neural probe shank flexibility will be useful. For the first time, we have successfully fabricated flexible neural probes with embedded microfluidic channels for dynamic control of neural probe stiffness by controlling fluidic pressure in the channels. The present hybrid neural probes consisted of polydimethylsiloxane (PDMS) and polyimide (PI) layers could provide the required stiffness for insertion and flexibility during operation. The PDMS channels were fabricated by reversal imprint using a silicon mold and bonded to a PI layer to form the embedded channels in the neural probe. The probe shape was patterned using an oxygen plasma generated by an inductively coupled plasma etching system. The critical buckling force of PDMS/PI neural probes could be tuned from 0.25–1.25 mN depending on the applied fluidic pressure in the microchannels and these probes were successfully inserted into a 0.6% agarose gel that mimicked the stiffness of the brain tissue. Polymer-based neural probes are typically more flexible than conventional metal wire-based probes, and they could potentially provide less tissue damage after implantation. Implantable neural probes are widely used to record and stimulate neural activities. These neural probes have been implanted into different tissues . Improving neural probe biocompatibility is one of the major tasks for many neural probe applications, especially for those that require long term usage. In recent years –5, biocoThe flexibility of the neural probes is related to the degree of tissue damage induced during brain micro motions after the probes are implanted. Neural probes with higher flexibility could cause less tissue damage . HoweverPrevious probe stiffening methods during insertion focused on using an extra piece of rigid structure to enhance the overall rigidity, rather than introducing dynamically controlled probe flexibility. In such cases, the neural probes cannot be reused after the bio-degradable layers were dissolved. Also, more tissue damage will be introduced due to the extra volume and stiffness of the rigid structure. Our present designs provide dynamic control of neural probe flexibility using adjustable fluidic pressure in the embedded channels. These neural probes were controlled to be rigid during insertion, and they returned to inherent flexibility after the insertion. The probes can be cleaned and reused easily. The neural probe flexibility was controlled by the probe materials, dimensions, and fluidic pressure in the embedded microchannels. In this work, neural probes were fabricated in PDMS, PI, and PDMS/PI hybrid substrates. The polymer-based neural probes are biocompatible and their mechanical properties can be optimized. The PDMS and PI polymers have different Young’s modulus, and they can provide different degrees of stiffness. Typically, PDMS is too soft for neural probe implantation into the brain tissue, while PI is more rigid compared to PDMS. A combination of PDMS and PI layers in neural probes could be tuned to reach the appropriate flexibility. For neural probe implantation, the fluidic pressure in the embedded microchannels of PDMS/PI neural probes could be adjusted to provide the needed stiffness. The fluidic pressure in the microchannels could then be released after implantation so that the neural probes will be highly flexible to reduce tissue damage. Probe flexibility for different designs as a function of fluidic pressure was simulated using the finite element method (FEM).Neural probes in PDMS were too flexible to be implanted into brain tissue, even with fluidic pressure in the embedded channels. The minimum critical buckling force for the PI probes was found to be 3.0 mN for 200 μm wide probes with 50 μm wide channels. This is higher than the required force of 0.5–1.0 mN , 31 to pIn this section, the designs, materials and fabrication technology for PDMS/PI hybrid probe were discussed. Moreover, the flexibility in terms of axial critical buckling force of these neural probes was measured using the buckling test that resembled the probe insertion into the targeted brain tissues. A smaller critical buckling force indicated the probe was more flexible.The dimension of fluidic channel needed to be large enough to provide a large range of flexibility control, and bonding area needed to be large enough to ensure there was no channel leakage when pressure was applied. Thus, probe widths of 200, 300, and 400 μm was selected in this study. The embedded microchannels formed a ‘U’ shape in the neural probe as shown in The fabrication of the hybrid PDMS/PI neural probe is shown in 2 plasma. After the photoresist was removed, the Si stamp was etched for an additional 40 μm to realize 60 μm total channel plate thickness (A deep reactive ion etching (DRIE)  system was used to etch the Si stamp in 2 steps. Initially, 20 μm Si was dry etched to define the channel depth and the remaining negative photoresist was removed using an Ohickness . The remhickness . A 60 μmhickness . Due to hickness .2 plasma (Plasma-Term 790 Series) using 25 sccm O2, 65 W stage power, and 700 mTorr chamber pressure for 20 s. The PDMS channel plate and the PI cover plate were bonded together with low pressure for 2 min, followed by baking at 130°C for 1 h  . Neural The critical buckling force was determined by measuring the force exerted by the neural probes on a microbalance  with resolution of 0.01mg. At the initial contact between the probe and the balance, the force value was set to zero. Two syringes filled with water were connected to the inlet and outlet located at the two ends of the U-shaped channel via a pressure sensor. Fluidic pressure was applied by fixing the plunger of one syringe, while slowly pushing the other plunger until the required pressure was reached . Fig 2E All numerical data was shown in average value with standard deviation. Data was compared by Student's t-test, and two groups of data were considered to have significant difference when p <0.05 .In this neural probe design, the U-shaped channel was filled with liquid. The total occupied space of the channel is the largest when there were no bending or other types of probe shank deformation. When the channel was bent under external force, this space would shrink accordingly. The fluidic pressure is an indicator of how well the channel is filled. A higher pressure indicated that the channel was filled with fluid that could stand against larger external force, thus more difficult for the probe to deform. In this case the overall stiffness of the probe became higher.in vivo operation with low tissue damage.The critical buckling forces for 60 μm thick PI-based neural probes with different probe width and channel width are shown in The fabricated probes were inserted into a 0.6% agarose gel that mimicked the brain tissue mechanical properties  as shownk) in Nm-1 was calculated for all PI and PDMS/PI hybrid probes without applying any pressure by the following equations:Ec is the Young’s modulus of the PDMS-PI composite (Pa), VPDMS and VPI are the material volume, w is the probe width (m), t is thickness (m) and l is the probe length (m). The calculated stiffness constants were 1.68×10−2 to 3.37×10−2 Nm-1 for PDMS/PI probes and 0.17 to 0.35 Nm-1 for PI probes. The stiffness constant of PDMS/PI probes with the smallest dimension was comparable to the k value of 7.1×10−3 Nm-1 in the literature that showed significantly reduced tissue damage and glial formation [In order to characterize the flexibility of the neural probes, PI probes with width of 200, 300, and 400 μm were fabricated. Stiffness constant  and copper (Cu) metal wire probes with a diameter of 70 and 150 μm, respectively, were used to compare the probes flexibility. The micrographs of the probe positions inside the gel at their starting and ending positions after the gel was moved laterally by 4 mm were compared . PI probThis work demonstrated the utilization of embedded microchannels in neural probes to dynamically control their stiffness in order to reduce motion induced tissue damage. By adjusting the polymer materials, neural probe dimensions, and fluidic pressure in the microchannels, probe stiffness could be dynamically controlled so that they could be inserted easily into the targeted tissue, but returned to be flexible after implantation by changing the fluidic pressure. Therefore, using the combination of PDMS and PI layers in the developed neural probes with embedded microchannels, they have the advantages of controlling the probes to be stiff enough for insertion with pressure applied in the channels and becoming more flexible after penetration by releasing the pressure to potentially reduce tissue damage.S1 Fig(a) Displacement of neural probe tips after gel was moved for 4 mm. (b) Displacement for neural probe tips with 200, 300, and 400 μm wide PI probes and 70 μm nickel/chromium (Ni/Cr) and 150 μm diameter copper (Cu) metal wire probes.(TIF)Click here for additional data file.S1 FileFlexible neural probes fabricated in PDMS/PI with microfluidic channels and electrodes. Critical buckling force of the probes was simulated and measured for various probe and channel dimensions, as well as different pressure in channel. Insertion force of neural probe in agarose gel and probe tip displacement were studied.(PDF)Click here for additional data file.


We present an alternative approach to calculate more accurate detection p values utilizing non-specific background fluorescence. We evaluate and compare our proposed approach of filtering observations with conventional ones by assessing the detection of Y-chromosome probes among males and females in 2755 samples from 17 studies on the 450K microarray and masking of large outliers between technical replicates and their impact downstream via an EWAS reanalysis.DNA methylation microarrays are popular for epigenome-wide association studies (EWAS), but spurious values complicate downstream analysis and threaten replication. Conventional cut-offs for detection n = 729).In contrast to conventional approaches, ours marks most Y-chromosome probes in females as undetected while removing a median of only 0.14% of the data per sample, catches more (30% vs. 6%) of large outliers , and helps to identify strong associations previously obfuscated by outliers between whole blood DNA methylation and chronological age in a well-powered EWAS (minfi package) was added to ewastools, an R package for comprehensive quality control of DNA methylation microarrays. Scripts to reproduce all analyses are available at doi.org/10.5281/zenodo.1443561.We provide guidance for filtering both 450K and EPIC microarrays as an essential preprocessing step to reduce spurious values. An implementation (including a function compatible with objects from the popular  Suggested p value cut-offs in the literature span several magnitudes from 0.05 to 1e−16 , although 0.05 and 0.01 are most commonly employed. Lehne et al. systematically evaluated detection p value cut-offs in a single large study based on the idea that probes targeting the Y-chromosome should be detected in samples from males but not females [p value cut-off choices, we demonstrate a modification to computing detection p values based on fluorescence resulting from non-specific binding that reflects the background noise distribution more accurately and thus further improves the sensitivity to aberrant values. We provide updated software and guidance for researchers conducting epigenome-wide association studies to easily incorporate this important preprocessing step in their analytical pipeline.The Illumina Infinium HumanMethylation450 (450K) and the more recent Infinium MethylationEPIC (EPIC) arrays are two widely popular platforms for epigenome-wide association studies (EWAS). Before beginning with downstream analyses, comprehensive quality control (QC) should be conducted to identify problematic samples. But samples passing QC still contain individual probes with spurious values that do not represent the underlying methylation state, for example, when targeted loci are present in low quantities due to amplification artifacts or are mutated and no longer match their intended complementary probe sequence. Affected probes feature mostly background noise and should be excluded. This decision is based on detection U) and methylated (M) variant of a CpG site, its methylation level in the DNA input can be inferred. The Infinium Type I probe design includes separate beads for U and M, whereas the Infinium Type II design combines U and M on the same beads but measured with different dyes/color channels [U probe features a very high intensity, whereas the M intensity is low, or vice versa, in the case of a completely methylated CpG site. Thus, the total fluorescence intensity T (the sum of U and M) for a given site is—to a certain degree—independent of the methylation level itself. High intensities usually indicate good signal-to-noise ratio and such probes are hence deemed detected, whereas low intensities consist mostly of background noise and thus are better classified as undetected probes. Where to draw the line is based on the concept of detection p values: the parameters of a normal background distribution B are estimated based on a set of probes thought to feature mainly background noise and a p value is computed using a z test. If the observed value of T is unlikely to be generated by B , a probe is considered detected, otherwise undetected. The previously recommended cut-off of 1e−16 by Lehne et al. is based on minfi v1.2.0 [B (adding standard deviations instead of variances of the U and M components of T).Treating DNA with bisulfite converts epigenetic modifications into distinct base sequences. In combination with subsequent whole-genome amplification, the ratio of methylated/unmethylated CpG sites is translated into differences in abundance of these distinct sequences. These hybridize to complementary probes on the 450K and EPIC microarrays and are subsequently linked with fluorescent dyes. By comparing fluorescence intensities of the probes targeting the unmethylated methylated CpG sites were identified by selecting the 1000 CpG sites with β values closest to each of the two peaks of the bimodal β value distribution  repository. Two thousand seven hundred fifty-five samples  were used to assess how detection ocessing .β values because of their linear relation with cell proportions. Proportions of seven major leukocyte types were estimated using the algorithm developed by Houseman et al. [Finally, we evaluated the impact on downstream analyses: a dataset of 732 peripheral blood samples with individuals aged 14 to 94 years from a population-based cohort .Code to reproduce all analyses is available at B using Illumina’s built-in negative control probes (NEG), we evaluated potential cut-off choices ranging from 1e00 to 1e−80, and for the higher non-specific fluorescence (NSP) intensities, we evaluated cut-off choices from 1e00 to 1e−03 as they resulted in higher detection p values. Among the 416 total Y-chromosome probes on the 450K array, the conventional and most widely used NEG/0.01 filter resulted in a median of 172 probes (41%) called detected among samples from females, whereas a NSP/0.01 filter resulted in a much lower median of 55 probes (13%) per sample. Among samples from males, the median number detected was 416 (100%) and 415 (100%) for NEG/0.01 and NSP/0.01, respectively. Figure When estimating Evaluating the proportion of times each Y-chromosome probe was detected  among all 1442 female samples revealed very distinctive patterns depending on the chosen filter  among the probes deemed detected in both index sample and replicate, and 9.5 pp (NEG/0.01) and 8.0 pp. (NSP/0.01) for all other pairs, respectively. Eleven thousand eight hundred seventy-seven probe pairs were large outliers with a difference > 20 pp, of which 743 (6%) (NEG/0.01) and 3602 (30%) (NSP/0.01) were classified as undetected in either sample, respectively.p values for multiple comparisons. Forty-four hits were found only for NEG/0.01 and 92 hits were found only for NSP/0.01 while 38,755 were common to both. Importantly, both approaches only differ in which observations are deemed undetected while β values of the retained observations are identical. Figure p values < 1.06e−07) only after applying NSP/0.01, demonstrating how even small numbers of spurious values included under current practices (NEG/0.01) contaminate the distribution of observed methylation β values and can obfuscate even strong associations, as in some instances the statistical significance jumps many orders of magnitude . The top nine of the 44 former hits, i.e., associations losing significance , are shown in Fig. Testing the association of 473,864 autosomal CpG sites with chronological age resulted in 38,799 hits for NEG/0.01 and 38,847 hits for NSP/0.01, significant at the 5% level, respectively, after Bonferroni adjustment of p values were so far based on a background noise distribution estimated from negative control probes, a recent paper proposes switching to out-of-band intensities [Background subtraction/correction is a common preprocessing step for 450K and EPIC data. Whereas some methods utilize negative control probes to estimate a background noise distribution, others employ so-called out-of-band intensities for this task . Yet botensities . Zhou etp values can not only protect against false-positive findings, but against false-negative findings as well.Similarly to Zhou et al., we propose swapping out negative control probes for another set of probes better suited to estimate the background noise distribution. However, we recommend against using out-of-band intensities as these, even though of Infinium Type I design, may equally suffer from cross talk  just likp values are therefore not accurate, which is why an extreme cut-off such as 1e−40 was necessary to achieve similarly stringent results as NSP/0.01. The NSP/0.01 filter also helped to exclude many more (30% vs. 6%) of the large outliers (> 20 pp) between technical replicates while dropping only a small fraction (0.14%) of the overall data. It has become common practice in the methylation microarray literature to drop probes and even entire samples if the number of measurements deemed undetected exceeds a certain threshold on the grounds that such probes/samples are unreliable [We recommend to utilize non-specific fluorescence to estimate the background distribution and a cut-off of 0.01 as this combination (NSP/0.01) classified most Y-chromosome probes in female samples as undetected while still calling almost all Y-chromosome probes among males. The fact that the conventional and widely used filter NEG/0.01 led to so many calls of Y-chromosome probes among females demonstrates that negative control probes do not faithfully reflect background noise levels and that derived detection reliable . Such exp value cut-off impacts statistical inference as unfiltered spurious values can obfuscate even strong associations with the discovery of weak associations presumably being even more impeded, supporting the need for more stringent data preprocessing. It should be noted that this conclusion holds regardless of whether the associations with chronological age discovered here are genuine or the result of residual confounding. However, the uncovering of new associations here comes with a tradeoff as a smaller number of sites lose significance. In some instances, this may include false positives previously driven by now discarded outliers, but other probes may decrease significance due to a reduced sample size. While our proposal represents an improvement and reasonable tradeoff over the current implementation of detection p values, there may be further room left to discriminate between reliable and unreliable observations.Although our method improves the detection of large outliers among technical replicates, the majority of large outliers still remained: this may indicate that there are other unknown mechanisms that create such spurious values besides low fluorescence. Most probes deemed undetected showed deviations between replicates smaller than 20 pp although their median absolute difference was still much larger than among detected probes indicating that they should be removed as imprecise measures. In our EWAS reanalysis, strong associations were revealed in probes which had a substantial proportion of observations dropped after filtering. Furthermore, with the exception of a few exposures such as smoking, effect sizes of epigenomic associations in whole blood are often very small and removing these least reliable data points may strengthen statistical power. DNA methylation microarray studies have found and validated associations with BMI , diabetep value function greatly decreases the prevalence of large outliers that can drive false-positive findings and can avoid false-negative findings. Our R package ewastools provides the necessary functions following our recommendations and is compatible with raw .idat files or minfi processing pipelines.A more stringent preprocessing of microarray DNA methylation data is required to filter out spurious values. We demonstrate that restricting to measurements that pass our new detection


Myo‐inositol trispyrophosphate (ITPP) is an allosteric effector that reduces the oxygen‐binding affinity of haemoglobin and facilitates the release of oxygen by red blood cells. We investigated herein the oxygenation effect of ITPP in six tumour models and its radiosensitizing effect in two of these models. The evolution of tumour pO2 upon ITPP administration was monitored on six models using 1.2 GHz Electron Paramagnetic Resonance (EPR) oximetry. The effect of ITPP on tumour perfusion was assessed by Hoechst staining and the oxygen consumption rate (OCR) in vitro was measured using 9.5 GHz EPR. The therapeutic effect of ITPP with and without RT was evaluated on rhabdomyosarcoma and 9L‐glioma rat models. ITPP enhanced tumour oxygenation in six models. The administration of 2 g/kg ITPP once daily for 2 days led to a tumour reoxygenation for at least 4 days. ITPP reduced the OCR in six cell lines but had no effect on tumour perfusion when tested on 9L‐gliomas. ITPP plus RT did not improve the outcome in rhabdomyosarcomas. In 9L‐gliomas, some of tumours receiving the combined treatment were cured while other tumours did not benefit from the treatment. ITPP increased oxygenation in six tumour models. A decrease in OCR could contribute to the decrease in tumour hypoxia. The association of RT with ITPP was beneficial for a few 9L‐gliomas but was absent in the rhabdomyosarcomas.Tumour hypoxia is a well‐established factor of resistance in radiation therapy (RT).  This compound has intrinsic anti‐cancer properties as it has demonstrated therapeutic efficacy in various animal models when used alone or combined with chemotherapies.Considering the compelling link between tumour hypoxia and treatment outcome, efforts have been made to develop effective hypoxia‐targeted therapies.22.1Four mouse tumour models  and two rat tumour models (rat 9L‐glioma in Fischer F344 rats and rat rhabdomyosarcoma in WAG/Rij rats) were used. The origin of cell lines and animals were reported elsewhere.2.22 measurements were carried out 15 minutes after the induction of the anaesthesia. The linewidth of the first‐derivative EPR spectrum that was the average of five 1‐minute scan accumulations was then converted to pO2 using a calibration curve.An L‐bandEPR spectrometer  operating at 1.2 GHz was used to evaluate the dynamic change in tumour oxygenation upon ITPP administration. Charcoal suspension , used as the oxygen sensor, was introduced intratumourally (about 60 µL for a mouse tumour and 200 µL for a rat tumour) 1 day before the experiment. The charcoal is dispersed over the whole tumour. During EPR recording, animals were anaesthetized with 2% isoflurane in air and their body temperature was maintained at 37 ± 1°C using a circulating warm water system. This anaesthesia regimen was previously demonstrated not to disturb the haemodynamics in rodents.2.3Hoechst 33342 (Sigma) staining was used to assess 9L‐glioma perfusion 1 day after completion of the treatment (2 g/kg ITPP once daily for 2 days). Rats were killed exactly 2 minutes after intravenous injection of Hoechst solution . Tumour fragments were rapidly excised, embedded in optimal cutting temperature compound and frozen in liquid nitrogen‐cooled isopentane. Frozen sections of 5 μm thickness were photographed using a Zeiss Mirax fluorescence microscope and images were analysed using Frida software. The percentage of tumour perfusion was calculated as the ratio of Hoechst‐positive area to the total area of tumour sections .2.415N‐PDT  as the oxygen sensor. Cells were incubated with 10 mmol/L ITPP for a period of 2 or 6 hours. LY294002 (Invitrogen), a PI3K inhibitor, was also included in the study at a concentration of 20 µmol/L to compare its effect with ITPP. After harvest, cells were resuspended in culture medium at a concentration of 107 cells/mL. About 100 µL of cell suspension was mixed with 100 µL of 20% dextran to avoid agglomeration and then sealed in a glass capillary tube in the presence of 0.2 mmol/L 15N‐PDT. Cells were maintained at 37°C during the acquisition of the spectra. EPR linewidth was measured every minute and reported on a calibration curve to obtain the oxygen concentration. OCR was determined by the slope of the decrease in oxygen concentration in the closed capillary tube over time.The impact of ITPP on OCR was assessed on six cell lines  using a Bruker EMX X‐band EPR spectrometer operating at 9.5 GHz and 2.5137Cs irradiator IBL‐637 , 20 Gy for rhabdomyosarcoma and 30 Gy for 9L‐glioma. Animals anaesthetized with isoflurane (2% in air) were placed on a plexiglass and protected from the beam through a lead block of 3 cm thickness while the tumours were exposed through a hole 25 mm in diameter. The animals were turned midway through the exposure time to enhance the uniformity of dose distribution. Irradiation doses were selected on the basis of their respective radiation sensitivity to ensure a significant growth delay in the irradiation group compared to the untreated one. Treatment effect was analysed based on tumour growth delay assay. Tumours were measured on the starting day of treatment to determine the initial size and then at least twice a week until the end‐point . Clonogenic assays were also performed to evaluate the radiosensitization effect of ITPP on rhadomyosarcoma and 9L‐glioma cell lines (The regimen of 2 g/kg ITPP once daily for 2 days was used. Irradiation was performed 2 hours after the second administration of ITPP (time at which tumour oxygenation was shown to be highest). Two rat models (9L‐glioma and rhabdomyosarcoma) were employed; rats were randomly divided into four groups: vehicle, ITPP, RT + vehicle and RT + ITPP. Single dose of irradiation was delivered by a ll lines .2.6t test or Mann‐Whitney test when data were not normally distributed. Log‐rank test was used to compare Kaplan‐Meier curves. P < 0.05 was considered statistically significant for all tests.All results were expressed as mean ± SEM. Differences between groups were analysed using 33.12 evolution upon ITPP administration was conducted on various tumour models to assess the effect of this compound on tumour oxygenation. The first test with 9L‐glioma and FSaII showed a significant increase in tumour pO2, quickly within 2 hours after the injection of a single dose of 2 g/kg ITPP   was obtained  was assessed on 9L‐glioma Figure . Areas o3.3To investigate the radiosensitization effect of ITPP, we combined the optimal treatment schedule of ITPP (2 g/kg once daily for 2 days) with irradiation of 20 and 30 Gy on rhabdomyosarcoma and 9L‐glioma, respectively. The results of tumour growth delay assay are presented on Figure 4Myo‐inositol trispyrophosphate has demonstrated therapeutic efficacy in a wide range of animal modelshttp://normoxys.com/clinical-trial-results/). Using the OxyLite system, Raykov et al found that a single ITPP injection induced an increase in partial pressure of oxygen for almost 1 week in pancreatic tumour xenografts.2 in vivo and is able to repeat the measurements at the same site over long periods of time. Our results obtained by EPR indicated that ITPP treatment quickly increased tumour oxygen levels in the six tested models and reached the maximum effect at 2 hours post‐administration. Such elevated pO2 was sustained for at least 1 day and then gradually decreased  would be the most appropriate choice to observe the modulation of tumour OCR by ITPP.Regarding the mechanism, ITPP is an allosteric effector that reduces the oxygen‐binding affinity of haemoglobin and thus facilitates the oxygen release by red blood cells.Finally, the anti‐tumour properties of ITPP were investigated on rhabdomyosarcoma and 9L‐glioma whose radiosensitivity has been shown to be correlated with oxygenation level.In summary, our data consistently demonstrated the increased tumour oxygenation in six animal models upon ITPP administration. We also showed in a proof of concept experiment that the enhancement in oxygen level likely resulted from a decrease in oxygen consumption rather than an increase in oxygen perfusion at least at the early stage. The increase in tumour oxygenation induced by ITPP only partly radiosensitized one of the two investigated models. Taking our finding together with the previous reports from the literature, ITPP possesses to some extent potential characteristics that can be beneficial to cancer treatment. However, to take full advantage of its capacity and to move further into clinical trials, a complete picture on the mode of action of this compound is mandatory. Future studies should focus on the underlying mechanism of ITPP and on how the oxygenation effect would be involved in the ultimate therapeutic efficacy of ITPP.The authors confirm that there are no conflicts of interest. Click here for additional data file.


In the original publication the grant number is incorrectly published. The correct grant number should be read as “17140901600”. The corrected contents are provided in this correction article. This work was partially supported by grants from the National Natural Science Foundation of China (Nos. 81670470 and 81600149), a grant from the Shanghai Municipal Commission for Science and Technology  and a grant from National Key Research and Development Program (2016YFC0905100).


Development of neurological complications of varicella zoster virus reactivation is relatively uncommon, particularly in an immunocompetent child.An 11-year-old Asian girl presented with headache and skin rash on her left chest. She was diagnosed with meningitis, and herpes zoster was confirmed by polymerase chain reaction using cerebrospinal fluid. Acyclovir was administered intravenously. Given the favorable evolution of the clinical course, she was discharged from the hospital on day 8 of her illness. She had no apparent sequelae or comorbidities at the time of the 6-week follow-up.Neurological complications such as meningitis due to varicella zoster virus reactivation are uncommon, especially in an immunocompetent child; no specific immune deficiency was identified in our patient. We conclude that, although rare, varicella zoster virus should be recognized as a potential cause of viral meningitis in immunocompetent children. Varicella zoster virus (VZV) is a member of the herpesvirus family and causes varicella (chickenpox). After primary infection, VZV establishes latency in the cranial nerve and dorsal root ganglia. Cell-mediated immunity to VZV declines in elderly people or in the immunosuppressive state, and it leads to virus reactivation that can cause herpes zoster. Development of neurological complications due to VZV reactivation is considered to be relatively uncommon, particularly in an immunocompetent child. In this report, we describe herpes zoster and meningitis in an immunocompetent girl.An 11-year-old Asian girl presented with headache and skin rash on the left side of her chest that had begun 3 days earlier. She had been diagnosed with varicella when she was 2 years old and therefore had no history of receiving the VZV vaccine. She did not have any episode associated with primary immunodeficiency.Before the onset of illness, she had been feeling fatigue due to exhaustive preparation for a school gymnastics event over the course of several weeks. She visited the local clinic due to repeated afebrile vomiting and severe headache. On the same day, she was admitted to our hospital with a concern of meningitis.Upon her admission, physical examination revealed a body temperature of 37.2 °C, respiratory rate of 20 breaths/min, heart rate of 85 beats/min, and normal hemodynamic parameters with blood pressure of 117/68 mmHg. She was noted to have a maculopapular rash evolving into vesicles with erythematous regions on the left side of her chest  examination revealed normal protein concentration (36 mg/dl), normal glucose level , and lymphocytic pleocytosis (429 lymphocytes/μl). Bacterial culture of CSF yielded no growth. Varicella zoster virus (VZV) deoxyribonucleic acid (DNA) was detected in CSF by polymerase chain reaction (PCR) on day 5. Results of blood examination were within normal ranges, including white blood cells (7180/μl), leukocytes (5220/μl), lymphocytes (1507/μl), monocytes (287/μl), eosinophils (43/μl), and basophils (28/μl). Results of VZV anticomplement immunofluorescence studies revealed values of 19 mg/dl for immunoglobulin G (IgG) and below 1 mg/dl for IgM, which indicates the previous infection and acquisition of humoral immunity against VZV. Moreover, there was no increase in the inflammatory biomarker levels. She had normal levels of quantitative immunoglobulins and lymphocyte markers: IgG 1106 mg/dl , IgA 71 mg/dl , IgM 132 mg/dl , CD3 71.6% , CD4 32.1% , CD8 38.8% , CD4/CD8 ratio 0.83 , CD19 12.8% , and CD56 15.5% .She was diagnosed with aseptic meningitis and cutaneous manifestation of herpes zoster despite immunocompetence. Acyclovir (45 mg/kg/day) was administered intravenously for 3 days from admission. Because the symptoms of headache, neck stiffness, and skin rash eventually resolved, treatment was switched to oral valacyclovir (75 mg/kg/day) for another 10 days. Given the favorable evolution of the illness, she was discharged from the hospital on day 8. She had no apparent sequelae or comorbidities at the time of the 6-week follow-up.Herpes zoster is caused by the reactivation of VZV that remains in the sensory ganglion cells after primary infection of varicella or vaccination. Waning of cell-mediated immunity to VZV declines in the elderly or in the immunosuppressive state; this is thought to trigger the reactivation of herpes zoster because antibody remains unchanged or even increases with age , 2. TherThe distribution of the age at onset of herpes zoster among immunocompetent and immunocompromised children was similar. Both had two peaks, namely at 4–5 years and 10–13 years of age [Development of neurological complications such as meningitis in varicella zoster is believed to be rare. In a study of 859 adult patients with herpes zoster, meningitis was reported in only 0.5% cases . AuthorsFor diagnosis, PCR analysis of viral particles in the CSF is crucial when aseptic meningitis is suspected. A rise in antibody titer between paired serum samples might also be helpful. Although dermatomal distribution of vesicular skin lesions is a typical symptom of varicella, an atypical case without any skin lesions has also been reported . Thus, VThe optimal therapy for meningitis with varicella zoster infection has not been determined yet. However, the guidelines issued by the Infectious Diseases Society of America recommend the administration of intravenous acyclovir at 10–15 mg/kg every 8 h for VZV encephalitis . In our No specific immune deficiency was identified in our patient from either her medical history or blood examinations, such as immunoglobulins and several lymphocyte markers. Currently, we have no data to explain the co-occurrence of VZV reactivation and meningitis in the immunocompetent child. Several reports have suggested that VZV infection in the first year of life could be a risk factor for herpes zoster , 12. LowIn conclusion, neurological complications such as meningitis due to VZV reactivation are uncommon, especially in an immunocompetent child. No specific immune deficiency was identified in our patient. It may not be too difficult for an expert to list VZV as a cause of meningitis in immunocompetent children; however, it might be tough for a young trainee because aseptic meningitis with VZV is rare. We conclude that, although rare, VZV should be recognized as a potential cause of viral meningitis in immunocompetent children.


In this paper, we present Low-Bandwidth Distributed Applications Framework (LDAF)—an application-aware gateway for communication-constrained Internet of things (IoT) devices. A modular approach facilitates connecting to existing cloud backend servers and managing message formats and APIs’ native application logic to meet the communication constraints of resource-limited end devices. We investigated options for positioning the LDAF server in fog computing architectures. We demonstrated the approach in three use cases: (i) a simple domain name system (DNS) query from the device to a DNS server, (ii) a complex interaction of a blockchain—based IoT device with a blockchain network, and (iii) difference based patching of binary (system) files at the IoT end devices. In a blockchain smart meter use case we effectively enabled decentralized applications (DApp) for devices that without our solution could not participate in a blockchain network. Employing the more efficient binary content encoding, we reduced the periodic traffic from 16 kB/s to ~1.1 kB/s, i.e., 7% of the initial traffic. With additional optimization of the application protocol in the gateway and message filtering, the periodic traffic was reduced to ~1% of the initial traffic, without any tradeoffs in the application’s functionality or security. Using a function of binary difference we managed to reduce the size of the communication traffic to the end device, at least when the binary patch was smaller than the patching file. The Internet of things (IoT) has become an enabling infrastructure for a wide range of novel applications. Traditional IoT architectures are cloud-centric , where aIoT end devices are frequently limited in their computational and storage resources or communication bandwidth, and operate under limited available energy. It is therefore unsurprising that there is a set of platforms, protocols, and messaging formats, as well as networks to support these stringent requirements. They range from efficient embedded IoT platforms  over adaGateways have long been an essential part of heterogeneous and distributed information, service and communication infrastructures  in 2/3G, serving (SGW) and packet data network (PGW) gateways in EPC, etc.). A communication gateway in IoT makes possible the interoperability of heterogeneous communication systems, but can also provide additional communication services, as for example security. An application gateway is finely tuned based on the given application’s requirements. If it is data driven, gateways focus on message and protocol adaptations and optimizations. They can, e.g., provide API harmonization. Application-driven ones assist mediating services  and adapFog and related mobile edge computing provide architectural approaches that are complementary to traditional cloud-centric systems . Thus coImprovements that IoT and blockchain integration can result in decentralization and scalability; device management and authorization; trustworthiness in data; trusted sharing service; data monetization; autonomous machine-to-machine transactions; including smart contract—based decentralized applications; and micropayments. Application opportunities are being sought in areas as smart cities, smart homes, mobility, health, logistic, and food traceability . There aThe research in  defined Our study was initially motivated by our previous work with blockchain IoT systems. We were focusing on the blockchain IoT client architectures  and the Beside the evident impact in reduction of communication traffic, the proposed solution maintains high flexibility and can be easily adapted to, e.g., changes in remote APIs, or can accommodate other/new communication and application protocols.Designed and implemented an application-aware gateway, LDAF, which can be a part of a fog computing architecture. It enables fog data services for reduction of communication traffic of bandwidth constrained IoT devices to connect them to arbitrary cloud APIs.We set-up and executed a decentralized application use case with blockchain-enabled IoT devices, where the LDAF gateway was applied and adapted to the application’s operation, to reduce the size and number of messages needed for the IoT application.For blockchain-enabled IoT devices we measured that the binary encoding of message content reduced the periodic traffic from 16 kB/s to ~1.1 kB/s, i.e., 7% of the initial traffic. Additional optimization of the application protocol in the gateway and message filtering further reduced the periodic traffic to ~1% of the initial traffic, without any tradeoffs in the application’s functionality or security. In two other supporting use cases we demonstrated the possible applicability of LDAF for other fog services.In our research we performed the following.In ® program, which allows Wi-Fi client devices to connect directly with each other by setting up ad-hoc networks, without going through a wireless access point or hotspot. In hybrid IoT–BC systems, PAC and Wi-Fi Direct can complement the IoT–BC interactions where low-latency or lager peer communication bandwidth is required.IoT device ecosystems impose changed and shifted priorities for end devices. IoT devices often require long battery life, low deployment and maintenance costs, long operation lifetime, and careful conservation of devices and network resources . These aThe IoT protocol stack demonstrates new or adapted transport and application protocols. The TCP/IP header and protocol redundancy can become an issue if low-bandwidth communications are sought. At the transport layer the simpler UDP can be applied instead of TCP. However, as it only provides a best-effort transport service, an adapted security protocol DTLS was needed to operate over UDP. DTLS  implemenThe application layers Constrained Application Protocol (CoAP)  and MessApart from the aforementioned CoAP and MQTT, additional application-layer protocols strengthen support for IoT devices . The OMA(i)Support IoT specific protocols in cloud APIs: not likely, if the cloud service is externally provided and the IoT solution developer therefore cannot affect the cloud API implementation and functionality.(ii)Develop a dedicated proxy function between the available API implementation and the one appropriate for our IoT devices.(iii)Apply a modular application gateway, as the LDAF presented in this paper, where common API and device connectivity functions are readily available and where the developers can concentrate on application-specific optimization of traffic volumes.Despite various adaptations of communication networks and protocol stacks for IoT, cloud-centric IoT systems apply more common communication, transport, and application technologies to make the cloud resources available to others. These cloud systems range from, e.g., IoT collection and analysis platforms , public API aggregation: the LDAF server adapts to specific external API requirements  and adapts it to a common message exchange between itself and the client.Efficient binary message serialization: the content of the messages between the client and the server is binary encoded using Protocol buffers   or similApplication adaptation: server can adapt the application logic, e.g., to reduce the number of messages passed to the client to further reduce the communication requirements. It can also take over a part of a device’s application execution , and thus not only reduces the communication bitrate, but the processing and storage requirements at the end device too. This allows for applications beyond simple aggregation of APIs.The Low-bandwidth Distributed Application Framework (LDAF)  is comprThe LDAF framework is characterized by modular architecture, as depicted in Without LDAF the device would connect directly to the external API, and would therefore have to comply with the protocols, syntax, semantics, and application logic imposed by the API. These might not be well adapted to the device constraints.A LDAF message is comprised of a header and payload. As can be seen in A message sequence is required to associate the responses with the appropriate request. However, it could be applied in the future to increase the reliability of message transfer at the application layer . In push messages from the server to the client the SequenceNumber is omitted because such a message is not a response to a specific request.The message type determines which schema must be applied to decode the binary message content. Both the message Type and the SequenceNumber are emitted to the available Services, so that the corresponding Service decodes and processes the message.There can be an arbitrary number of message types required by one Service. This would make static type assignments inefficient; we therefore applied a dynamic assignment. The message type is indicated by the value at the corresponding location in the Type parameter. The Type parameter is interpreted by a service offset, which points to the first message type for each particular service. The following service offset points to the location after the last message type of the previous service. The offsets of all services applied in a connection are specified in the service definition.We thus minimized the data required for the appropriate type identification. For each message we thus introduced 2 B of additional overhead and only 1 B for push messages. The remainder is the payload. With 1 B for the Type field we can organize, e.g., 1 Service with up to 256 different messages types or two Services, one with 20 and the other with up to 236 types. For the Ethereum—based smart grid meter, for example, four types of messages are required: (i) a periodic block notification, (ii) an event notification, (iii) a request, and (iv) a response for transaction verification.LDAF messages are thus encoded in two substages. The first is the efficient binary serialization of the message content , HTTP (over TCP/IP) and integration with the LoRaWAN and The Things Network . In a siA connection receives and forwards messages from a device to the corresponding Service. During the connection setup, service definitions at the LDAF client and at the LDAF server are compared. If they do not match , the connection is not established. This assures that the server can provide all the required services, that the message types are correctly interpreted, and that the message payload is correctly decoded.Transcoding of messages: The service receives messages from the external APIs in format and protocol imposed by the API and transcodes the payload to the LDAF message format described in Application adaptation: The service in this case actively modifies the application logic. This affects the number of messages and further reduces the traffic for the end device. The service can determine whether a particular message is actually required for the device application and filter it out. It can, e.g., aggregate duplicated messages, negotiate and confirm message exchange with the API, and only pass the success notification to the end device, or build an aggregated response for the device that includes data collected from various APIs.Services address the needs of end device applications and therefore affect the size and/or number of messages for the end device. Two levels of application adaption are possible:Application adaptation helps LDAF servers distribute the application logic among the end device, cloud services, and the edge of the network.The LDAF framework is comprised of server and client entities. The LDAF server embodies the establishment and management of the end device connections, message encoding, service execution, and connections to external APIs. The LDAF client applies a set of client libraries for the LDAF server connections and message encoding and enables end device application logic to communicate with the framework.When the LDAF server is initialized, the Service.js library scans the subdirectory where all available services are defined and implemented. A particular service is usually implemented as a main library (main.js), with the specific application logic and a service definition file (serviceDef.js). Service definition files specify message type object. It also specifies (binary) message-content encoding mechanism, including corresponding message definition files . Upon initialization, connections to the external APIs are established, too.We applied two message types for the Ethereum—based smart-grid switch and counter. As seen in When a new client connects to a selected service on an LDAF server, they compare client and server service definitions, which must match to ensure correct encoding and decoding of messages. Both service definition files (serviceDef.js) and message definition files  must be present on the client’s side, too. We provide a generic client library (Client.js), which establishes the connection to the server and receives, transcodes, and transmits messages.(i)Function reachable through the network and implemented at  application servers, which are only connected, but not really integrated in the network infrastructure, and(ii)Function provided by the network, where LDAF is a functional component of, e.g., the 4G/5G network.There are several options for deployment of LDAF server in current and future communication networks. These options differ in their level of network integration. They can range fromApplying LDAF as an additional application server reachable through the network is a straightforward approach. It is independent from the underlying network infrastructure. The network is merely providing internet connectivity from end devices to the LDAF proxy. In LoRaWAN the application servers are already foreseen in the architecture. We tested this approach with a LoRaWAN TTN platform. In a similar manner the current 4G and emerging 5G networks provide channels to external internet servers and applications. This approach however, cannot fully exploit the possibilities that would appeared if LDAF were integrated into the network infrastructure.(i)Proximity of computational and storage resources to reduce latency and backend traffic.(ii)Collaborative sensing where LDAF would implement initial data preprocessing and aggregation of messages or streams from several end devices.(iii)Switch to non-IP data delivery for the end device for further traffic optimization.(iv)Information about the status of the radio-access network (RAN) for dynamic LDAF services or near-real-time application-aware performance optimization of the LDAF services.(v)Context awareness of the services, where context can reach beyond traditional cookie or location—based adaptations.The benefits of integrating LDAF in the network infrastructure—and thus the gateway being a function provided by the network—may include the following.LoRaWAN network architecture is typically laid out in a star-of-stars topology, in which the gateway is a transparent bridge relaying messages between end devices and a central network server in the backend. Gateways are connected to the network server (NS) via standard IP connections, while end devices use single-hop wireless communication to one or many gateways. The network’s server hosts all the intelligence; it manages packets from different gateways and sends them to the specific application server. In LoRaWAN, LDAF could become a part of the network server. A similar approach was taken in , where tIn mobile edge computing (MEC) , EvolvedThe LDAF proxy could be integrated in wired power-line communication systems (PLC), which are important customer–domain infrastructure in smart grid systems. The predominant use of PLCat the moment is remote meter reading; however, new smart grid applications envisage new communication over PLC. A PLC modem is integrated in the (smart) electricity meter on the customer’s side. The PLC communication is terminated in a message-aggregating gateway, located at the secondary substation. A secondary substation usually has its own local area network and wired broadband connectivity towards the higher levels of the grid’s architecture. The PLC in such a setup only provides low data rates, which limits the frequency of meter reading and practically narrows the scope of new associated services. An LDAF server would be placed at the message-aggregating gateway and the LDAF client in smart meters.To demonstrate the applicability of our approach we set up three use cases and applied the LDAF framework to manage the traffic from/to a communication-constrained device. The first case is a simple DNS query in which a device resolves a given host name to a corresponding IP address.The second example represents a realistic case of an IoT device, controlled over the Ethereum network. For this we applied our previously developed prototype system, dubbed Swether . This isThe third case targets binary transfers from the network to the IoT end devices, as, for example, in software updates and patching. To reduce unnecessary traffic to the device, we implemented a service that, instead of forwarding a complete new binary file to a device, calculates and forwards only the binary difference between the old and new version of the file. As in other cases, the originating file server does not implement such a service and only provides full versions of files. We can apply such a service with Swether, too, e.g., for system updates.To execute and evaluate the described use cases, we set up the environment depicted in In the blockchain use case, the end devices were five physical instances of the Swether device, as well as several emulated instances. For emulation only the specific sensing and actuation hardware was eliminated, otherwise they ran exactly the same application software as the Swether, and thus producing the same network load. The emulated instances were applied to create a number of concurrent clients while estimating scalability of the system. Beside the LDAF server, in the BC use case another server was applied, to host the Ethereum geth client. It had permanent Internet connection to keep the geth client synchronized with public the Ethereum network. For DNS and binary transfers to the IoT devices, corresponding HTTP request were launched from the end devices to the LDAF server, when needed.The entire communication traffic from/to LDAF server was captured and analyzed with Wireshark.During the operation we estimated the system requirements imposed to the LDAF server. Because the CPU and memory requirements were so low, we installed the LDAF server on a Raspberry Pi (RPi) 3 B as well. We tested it with up to 100 concurrent clients using the blockchain service in LDAF. The memory consumption was independent from the number of clients and was ~5%, i.e., 50 MB. The CPU load was varying. During the block processing (the Ethereum network creates a new block approximately every 20 s) the 1 s average was approximately 3–6%, otherwise ~1%. LDAF service implementation assures that the number of clients subscribed to the same service, does not significantly affect the CPU load. We would like to point out that a systematic resource monitoring of the server resources was not our research objective. The observed values can serve as indication and are application-specific and indication and are application-specific.We developed a new service definition (dnsServiceDef.js) and a simple DNS client. The LDAF client initiates a WebSocket connection for the transaction between the client and the LDAF server. The LDAF server sends a DNS query to the predefined DNS server and receives the responses. These transactions are encapsulated in the UDP messages. The service definition includes the parsing rules from the DNS response of the DNS server and forms message syntax for the LDAF client.The regular DNS transaction executed by the NodeJS environment results in one request and one response, as shown in The sizes of the messages in regular DNS transactions and in LDAF are comparable. This is because DNS is not a communication-intensive protocol. It applies UDP for transport and therefore gains an advantage of 12 B per packet over WebSocket using TCP. However, if we focus only on data fields in both transactions, the difference is more evident. Binary encoding in LDAF reduces the size of the message content field of all messages to about 1/5.Binary message content serialization by applying binary encoding of message content instead of textual (JSON). In this case all the messages from the Ethereum node are passed to the Swether. Bandwidth reduction is a consequence of more efficient encoding.Application adaptation reduces the number of block notifications that are periodically sent from the Ethereum node to the connected devices. For Swether, we applied this information to check the block depth for security reasons. After receiving an event notification for an action to be executed in the Swether, it first waits for a predefined number of blocks to verify that the notification is actually placed deep enough in the chain. However, in most other cases the block number notification is not required by the end device.We developed a new service definition (serviceDef.js) for LDAF server and the client was a Swether device configured to operate over LDAF. Upon service initiation the LDAF server connects to the remote Ethereum node. Just as in case of DNS, also here the client initiates a WebSocket connection for communication between the client and the LDAF server. The LDAF in our case enables two optimization scenarios:We therefore implemented block notification filtering at the LDAF (with an arbitrary filtering ratio), to reduce the number of messages. To maintain the application’s security, event validation was implemented in the LDAF service, too. Only verified event notifications are passed to the Swether. As Swether does not have to verify them, block number notification is no longer essential data for application operation. Only occasional block number notifications are applied to check the node synchronization.For the first scenario we set the LDAF service to pass every (1 out of 1) block notification to the Swether. We captured all the traffic from and to the LDAF server, so as to include the WebSocket session setup and at least 10 consecutive block notifications. The traffic between LDAF server and Swether was 1104 B and between the LDAF server and the Ethereum node about 16 KB. Due to binary message content, encoding the traffic was thus reduced to ~7% of that from the original API of the Ethereum node. For DNS, this advantage was minimal due to the small DNS query sizes and UDP. For Ethereum the reduction is substantial, as the traffic is reduced nearly to 1/14.However, the traffic was even more reduced when the application adaptation was applied. As shown in We developed a new service definition (serviceDef.js) for LDAF based on the bsdiff-nodejs library  to enablApplication adaptation. The LDAF server caches a version of a previously downloaded file. Upon request from the device or a push from the cloud server for an update, the LDAF service calculates the binary difference between the new and previous content and returns it to the device. The device patches the old version of the file.Despite binary content, binary LDAF message content serialization still takes place. It is a generic part of LDAF message encoding and not a part of the service. However, it does not additionally reduce the size of the already compressed binary content. If LDAF was applied exclusively for transferring binary files, the binary difference could be implemented as the default LDAF message content serialization. However, being a service allows for the broader use of the framework.The effect of this service predominantly depends on the binary difference of the two files transferred. To test the operation and estimate the impact of message and communication overhead, we used two binary installation files, named v62 and v63, each ~1.8 MB. The size of the binary difference patch to upgrade from v62 to v63 (as reported by bsdiff) was 1531 kB. The size of the patch when the v62 file was compared to itself was 145 B, to allow for the mandatory bsdiff patch header. Transferring the v63 file from the file server to the LDAF server resulted in 1942 kB in download. The corresponding patch from the LDAF server to the end device resulted in 1627 kB download. The difference between the original file v63 and the patch was 292 kB. The difference in download traffic was even a bit bigger, 315 kB, as a consequence of more efficient WS download compared to HTTPS. The void patch for v62 to v62 resulted in only 619 B download, and that was mostly due to WebSocket establishment. The exact communication traffic loads might vary a little bit, if the experiment was repeated, due to changed network conditions .Other file pairs would result in larger or smaller binary differences, but the key is that the LDAF framework reduces the size of the communication traffic at least as much as the binary patch is smaller than the patching file.We designed, developed, and tested an application-aware gateway framework for constrained devices—the LDAF. It facilitates flexible implementation proxying services to reduce the communication loads by efficiently encoding binary message content and adapting application protocol operation to affect the number of exchanged messages; it further reduces the traffic for the end device. The implementation proved to be robust, able to run several concurrent services and serving various clients.Currently we are implementing LDAF into a smart city ecosystem (EkoSMART). A part of this project is an integration platform, which supports a variety of smart city applications, including e-health, well-being, and smart mobility. These subsystems contribute IoT measurements into the integration platform and share it to various cross-domain applications. LDAF will enable the connection of communication-constrained devices and API integration.Our further research is focused on implementing an LDAF server on a LoRaWAN network server for application services that are aware of the network status.The next foreseen use cases are university e-learning and study management platforms. These platforms are independent, yet they must be synchronized. The available REST APIs, however, are not flexible enough to reduce the amount of traffic required to synchronize the two. This is not an IoT case and the traffic volumes are expected to be substantially higher. At the moment, ~15 MB of data is transferred for periodic syncing, although only ~1 MB is actually required. With LDAF we expect to lower the communication and processing during one synchronization, and thus enable more frequent synchronizations.


In the present work, an integrated optofluidic chip for fluid viscosity measurements in the range from 1 mPa·s to 100 mPa·s is proposed. The device allows the use of small sample volumes (<1 µL) and the measurement of viscosity as a function of temperature. Thanks to the precise control of the force exerted on dielectric spheres by optical beams, the viscosity of fluids is assessed by comparing the experimentally observed movement of dielectric beads produced by the optical forces with that expected by numerical calculations. The chip and the developed technique are validated by analyzing several fluids, such as Milli-Q water, ethanol and water–glycerol mixtures. The results show a good agreement between the experimental values and those reported in the literature. The extremely reduced volume of the sample required and the high flexibility of this technique make it a good candidate for measuring a wide range of viscosity values as well as for the analysis of nonlinear viscosity in complex fluids. Microrheology recently emerged among the techniques used to characterize the mechanical properties of soft materials or fluids, thanks to the possibility to study the response of samples at the molecular level. In addition, the capability to use small sample volumes (few µL or even less) also opened the way to the application of these techniques to expensive/limited materials, such as DNA-based samples ,2. The rIn the present work, we present a glass-based microfluidic chip suitable for measuring the viscosity of a fluid even with a sample volume <1 µL. This is achieved thanks to the force applied by two optical beams, propagating through the chip microchannel, on microbeads inserted in the sample and used as a probe. This force permits a precise micro-manipulation of the bead, allowing the study of its motion and hence the extraction of the fluid viscosity.The complete experimental procedure implemented to collect the data is explained in Optical forces provide a contactless and precise way to manipulate microbeads suspended in a fluid; hence optical tweezers have already been used to measure complex fluid viscosity as reported in ,8,9. In The trapping capability can be used to capture a single microbead flowing in the channel, but the optical beams also allow the movement of the bead across the transverse dimension of the microchannel by unbalancing the power of one of the two beams, thus shifting the equilibrium-position of the bead along the beam propagation axis.The forces exerted on the microbead by the laser beams have already been studied in detail elsewhere ,18. As nP) associated to the optical ray, on the reflection coefficient at the medium–particle interface (R) and on the incidence and transmittance angles  were introduced in the different fluids included in this study: Milli-Q water, ethanol, and water–glycerol mixtures.P , and its viscosity varies significantly with temperature. We thus created mixtures of glycerol with Milli-Q water, at different mass concentration, and we studied them at different temperatures.The obtained viscosity values, which are displayed in With the same temperature change, the glycerol mixture at higher concentration shows much larger variation, especially for 70% and 85% concentration. It should be mentioned that at higher concentration of glycerol, a higher laser power is required to have a full trajectory along the whole channel width compared to the lower concentration cases. Moreover, to avoid the laser heating effect when the fluid temperature is changed and consequent viscosity variation in the area near the waveguide, the initial part of the trajectory (about 20 µm) is discarded; this does not affect the viscosity retrieval process because of the non-inertial behavior in microfluidic condition, as stated in In conclusion, an integrated optofluidic chip is presented in this work that allows the measurement of fluid viscosity, and very low fluid sample consumption is achieved by using micropipette injection. By exploiting optical forces, the chip allows microbeads inside a fluid to be precisely controlled and manipulated, giving the possibility to assess fluid viscosity. The chip is also equipped with a temperature control, to maintain the fluid at the selected temperature. The fluid viscosity is derived by fitting the microbead optical shooting trajectory with theoretical curves. By real-time analysis, the shooting measurement can be repeated with the same bead in the same position of the fluid and with different beads in different positions of the sample, offering very high reliability and consistency. The chip has been successfully applied to measure the viscosity of several fluids and the obtained results well match the data already reported in the scientific literature. Additionally, the measurement range of fluid viscosity demonstrated in this study is very large thanks to the simple optical force control permitted by a simple variation of the laser power. However, higher laser power may introduce a laser heating effect and this can be evaluated and avoided by shooting trajectory shifting and partial trimming.The proposed chip and the technique reported here can also be directly applied to test the viscosity of fluids exhibiting more complex mechanical behaviors, as in the case of non-Newtonian fluids. It is interesting to notice that if non-Newtonian fluids are considered, a non-linear relationship between  samples .


S) as predicted by the elastohydrodynamic-lubrication theory (EHD).Under favourable conditions (depending on the fluid viscosity (η), co-seismic slip-rate (V), fault geometry (L/H02) and earthquake nucleation depth (∝σeff)), EHD might be an effective weakening mechanism during natural and induced earthquakes. However, at seismic slip-rate, the slip weakening distance (Dc) increases markedly for a range of fluid viscosities expected in the Earth, potentially favouring slow-slip rather than rupture propagation for small to moderate earthquakes.Fluids are pervasive in fault zones cutting the Earth's crust; however, the effect of fluid viscosity on fault mechanics is mainly conjectured by theoretical models. We present friction experiments performed on both dry and fluid-permeated silicate and carbonate bearing-rocks, at normal effective stresses up to 20 MPa, with a slip-rate ranging between 10 μm/s and 1 m/s. Four different fluid viscosities were tested. We show that both static and dynamic friction coefficients decrease with viscosity and that dynamic friction depends on the dimensionless Sommerfeld number ( The effect of fluid viscosity on fault mechanics is mainly conjectured by theoretical models. Here, the authors present experimental data from rock friction experiments, showing both static and dynamic friction coefficients to decrease with viscosity and dynamic friction to depend on the Sommerfeld number. Elastohydrodynamic lubrication (EHD), or the weakening induced by overpressure generated by the shearing of a thin viscous fluid between two subparallel and rough surfaces4, has been recognised for a long time in industrial processes6. EHD has also been invoked to explain the dramatic reduction of friction during earthquake slip in the presence of fluids8. However, up to date, the possible triggering of EHD in natural faults relies on theoretical models only, and was not tested at deformation conditions typical of seismic faulting. Furthermore, the experimental studies investigating fluid-rock interaction almost exclusively considered water11 as the fluid at both subseismic and seismic slip-rates12.Fluids with variable composition , rheology and physical state are pervasive within active tectonic faults. The viscosity of such fluids may vary over seven orders of magnitude, from 1013, which represent only a fraction of the total fault area, and can be filled by fluids and gouges. During fault sliding and depending on how the normal stress is partitioned between the asperities and the fluid, three different regimes can been distinguished14: the boundary lubrication regime (BL), where the normal stress is supported by solid–solid contacts; the fully lubricated regime (EHD), where the normal stress is supported by interstitial fluid; and the mixed lubrication regime (ML), where the normal stress is supported both by the solid–solid contacts and the fluid. The transition between these three regimes is controlled by the Sommerfeld number4V is the slip-rate, η is the lubricant dynamic viscosity at the estimated mean temperature14 of the slipping zone , L is the characteristic length over which the fluid pressure changes (related to the wavelength of the asperities), H0 is the initial average gap between the asperities (related to the height of the asperities) and σeff is the effective normal stress .For sake of simplicity, tectonic faults can be described at seismogenic depths as rough surfaces separating two solids, which are in contact at a number of asperities16 provided evidence of the influence of fluid viscosity by performing laboratory stick-slip experiments as an analogue of seismic events17. The setup consisted of poly-methylmethacrylate (PMMA) slabs lubricated by a film of viscous fluid (silicone and hydrocarbon oils). The presence of the fluid resulted in a smaller static friction with respect to that under room-humidity conditions. However, the fracture energy  increased in the presence of the fluid and was independent of the lubricant viscosity but dependent of the lubricant composition.Recently, Bayart et al.18 and propagation (mm/s to m/s)20 and, in general, at slip-rates at which the ML and the EHD regimes might be activated.Here, based on experimental and geological evidence, we discuss the effect of the viscosity of a fluid sandwiched between two rock slip surfaces at slip-rates characteristic of either earthquake nucleation (slip-rate from μm/s to mm/s)21  on full cylinders (diameter D = 50 mm) of Westerly Granite and Carrara Marble either in the presence of a liquid lubricant or under room-humidity conditions. Pore fluid experiments were performed under drained conditions (a membrane pump maintained a constant fluid pressure of Pf = 3 MPa), preventing macroscopic fluid pressurisation inside the vessel9. The experiments were performed by imposing a slip acceleration of 6.5 m s−2 to the samples up to a target slip-rate (V), followed by deceleration to V = 0 m/s. The target V ranged from 0.01 to 1000 mm/s, slip distance (U) from 0.1 to 4 m and imposed (σn) were up to 23 MPa (Supplementary Table 22), which limited fluid diffusion through the rock matrix out of the sliding surface during the experiments, (2) of their very small grain size and homogeneity and (3) because typical rocks of the seismogenic continental crust23.We performed 36 experiments with the rotary machine SHIVAH0 ~ RMS = 7–13 μm, for Carrara Marble and Westerly Granite, respectively, measured with optical 3D profilometer, Contour GT-I 3D-Optical Microscope, Bruker). We consider the perimeter of our sample (0.157 m) as the maximum characteristic wavelength , due to the periodicity of rotation of the rock cylinders.The sliding surfaces of all the samples were roughened by using 120 SiC abrasive paper  = 0.5 × τ(t) × V, where r is the radial distance from the centre of the sample, τ is the shear stress, was applied as a Neumann boundary condition24 to the edge of the model, which simulates the slip surface. Then, the estimated temperature at r = 2/3 R where R = 25 mm is the external radius of the sample, was used to correct the fluid viscosity following the empirical formula proposed by Cheng25 and to compute S  is a Newtonian, water-soluble, colourless fluid, which is stable under most conditions due to its high flash and fire points , which prevents phase changes and fire hazards when exposed to high temperatures due to frictional heating. The viscosities at room temperature were 1.002 mPa s (pure distilled water), 10.8 mPa s (60 wt% glycerol), 109.2 mPa s (85 wt% glycerol), and 1226.6 mPa s (99.9% pure glycerol). The temperature increase with increasing slip due to frictional heating during the experiments is expected to lower the lubricant viscosity. The increase in the bulk temperature on the shearing surfaces was estimated by using a 2D time-dependent model for heat diffusion. We assumed that all the mechanical energy was converted into heat  and we neglected heat losses by radiation and fluid convection (the liquid was inside a vessel). As a consequence, half the instantaneous heat flow-rate μ) is the ratio of the shear stress to the effective normal stress. For both rock types, at the beginning of the experiments, the shear stress acting on the fault increased until the static friction coefficient μstatic was overcome and slip initiated  until a peak friction μpeak was achieved at V ~ 1 mm/s when the target V was higher than 1 mm/s. Subsequently, μ decreased with slip to a minimum and approximately steady-state dynamic friction coefficient (μdyn) during a transient slip weakening phase over a slip distance (Dc)26  increased with V. We studied the dependency of μstatic and μpeak with η and S, respectively, for all the experiments  over the entire range of S 26 Fig. . In our a s Fig. . Insteadf S Fig. , had no μdyn varied with S ; at 10−3 < S < 1, μdyn decayed with S from ~0.7(S = 10−3) to ~0.2 (S = 1) (ML regime); at S > 1, μdyn slightly increased with increasing slip-rate (EHD regime). This Stribeck-type behaviour was similar for both water and water/glycerol mixtures but shifted to greater S values for the latter  or Carrara Marble (temperature and grain-size dependent29). Note that μdyn slightly decreased with increasing V as proposed by Di Toro et al.2, but was high scattered  is the Coulomb friction coefficient corresponding to μstatic under dry conditions , μc (=0.1) is the friction coefficient at the full film lubrication condition (~μdyn during EHD), and β and γ are empirically determined linear coefficients. The second and the third right-hand terms of Eq. (V) and the viscous friction (the increase in the friction coefficient due to the presence of viscous fluids). We used a linear regression procedure to estimate the coefficients β and γ for the for the original experimental dataset presented here   tested at seismic slip conditions, and where frictional melting occurred, highlighting the universality of EHD mechanism when fluids are present in the fault slipping zone , μc =0. is the f), μc =0. is the fV = 1 m/s under room-humidity and water conditions (corresponding to S < 1), showed evidence of frictional melting34. Instead, there was no evidence of frictional melting in the slipping zones recovered from the experiments conducted on 85 and 99% of glycerol where the fully lubricated regime (S > 1) was achieved  grains. Instead, there were no slipping zones made of submicrometre in size grains after the experiments conducted with 99% glycerol and at high slip-rate where the fully lubricated regime (S > 1) was achieved  , at least in the BL and ML regimes (S > 1)  Fig. ; however 1) Fig. . Indeed,τ0 is the initial shear stress on the fault (whose upper bound is τstatic = μstaticσeff), τdyn = μdynσeff), and where the S dependence of τdyn(S) and Gc(S) is made explicit. As seen in the left-hand term, the effectiveness of lubrication increases with the amount of fault slip U. A large value of Gc may prevent the dynamic propagation of smaller earthquakes, but as U increases the lubrication effect will become dominant. Because fracture energy will remain constant after the critical slip distance U = Dc  which control S, the same parameters are poorly known in nature. Here, below we attempt an estimate of the possible ranges of  for natural faults. Water is pervasive in the Earth's crust37: the viscosity of water ranges from ~1 mPa s at subsurface conditions (~1 km depth) to ~0.1 mPa s at a depth of 10 km  of the seismogenic continental faults, fine grain gouge material mixed with water may also act as lubricant whose viscosity depends on the gouge grain size and the solid volume fraction. Viscosities of ~10 Pa s have been estimated for such fluid-saturated gouges40. Moreover, fluids commonly used in hydraulic fracturing operations in engineering reservoirs have viscosities ranging between 1 mPa s and 1 Pa s. As a result, the viscosity of fluids in the upper crust is estimated to range from 0.1 mPa s to more than 10 Pa s.While our experimental protocol allows us to impose and estimate the parameters (20 as indicative of fault seismic slip-rate. The L parameter in S under EHD conditions corresponds to the longest (and dominant) wavelength in the mismatch between two rough sliding surfaces. In natural faults, L corresponds to the asperity wavelength, the longest of which is proportional to slip4 (and the magnitude) during a given earthquake. For earthquakes of magnitude ranging between 1 and 8, the average seismic slip and thus L = 1 mm–4 m41. Natural faults' surface roughness is almost self-similar42 with a ratio H0 is ranging between 0.001 to 4 mm for a M1 earthquake and M8 earthquake respectively. The lithostatic stress σ1 = ρrgz  minus the hydrostatic water pressure (with ρw = 1000 kg/m3) at the earthquake hypocentral depth yields as an indicative value of σeff = (ρr − ρw)g z/K, with ϕ = arctan(μ), μ = 0.75, acting on the fault; this results in σeff ranging from ~50 MPa (at z = 3 km) to ~170 MPa (at z = 10 km).A value of 1 m/s is widely acceptedV = 4 m/s, L = 1 mm–4 m, H0~0.001–4 mm, σeff ≤ 170 Mpa, η ofwater) would results in S values ranging from 5.0 × 10−3 to 1.0 × 10−6 at z = 10 km for Mw = 1 to Mw = 8 and from 70 to 0.07 at z ~ 0 km for Mw = 1 to Mw = 8  slightly reduce the static friction coefficient fostering fault reactivation and (2) trigger EHD lubrication during seismic slip. However, in our experimental results, for a large range of possible viscosity values of fault-permeating fluids, typical seismic slip-rates would result in intermediate values of the Sommerfeld number (10−3 < S < 1) and relatively large Dc values. As a consequence the fracture energy during seismic propagation of small events (U < Dc) is relatively large, inhibiting seismic rupture propagation in favour of slow strain energy relaxation by stable creep (S and Dc would both decrease at low slip-rate). In contrast, the presence of increasingly viscous fluids decreases the fracture energy dissipated for large events, making the fault weaker. Therefore, our results suggest that in the presence of highly viscous fluids, rupture is expected to grow quasi-statically on the fault until the slip of the order of tens of cm is attained (until the distance Dc is overcome). While the effect of fluid pressure in earthquake rupture has been previously explored mostly in terms of thermal pressurisation and effective stress, we argue that the role of fluid viscosity is also important in understanding the dynamics of a lubricated fault system, with implications for rupture energy budget and dynamic weakening of both natural and man-induced earthquakes.We have experimentally demonstrated that fluid pressure and 21, a rotary shear machines installed in Rome, on full cylinder samples of Westerly granite and Carrara marble. Target slip-rates (V) ranged from 10 µm/s up to 3 m/s, acceleration and deceleration ramps were imposed to 6.5 m/s2 and normal stresses were up to σn = 22.6 MPa. Mechanical data  were acquired at a frequency between 250 Hz and 25 kHz, depending on the target slip-rate. Slip, slip-rate and shear stress were determined using the method outlined in Niemeijer et al.43 and Tsutsumi and Shimamoto44. All the experiments μ vs. displacement for four different experiments performed with SHIVA under 10 MPa of effective normal stress (σeff), in presence of different mixtures of distilled water and glycerol in order to have an initial viscosity η0 increasing of a factor ca. 10, with a fluid pressure Pf = 2.7 MPa. In the figure, the apparent friction coefficient is fitted following the exponential decay function proposed by Mizoguchi et al.26Dc.In Supplementary Figure The 3D arithmetic surface roughness was determined on the sliding surface of 20 or 25 mm in diameter cores of the starting samples and post-mortem samples of Westerly granite and Carrara Marble using an optical profilometer ContourGT-I 3D Optical Microscope, Bruker Nano surfaces Division. The scan of the surfaces was performed imposing an overlap of 20% between two adjacent areas. RMS (root-mean-square) and the stitched images of the samples surface of Westerly Granite after the experiments performed under room humidity condition and in presence of the four different fluids at slip-rate of 1 m/s are shown in the Supplementary Figure S ≥ 1 , but slided at different slip-rate, had roughness of the same order of magnitude when S was higher than 1, the RMS of the samples at the end of the experiments was of the same order of magnitude of the initial RMS. In the experiments performed at high slip-rate the presence of high-viscous lubricants prevented the formation of melt and debris on the surfaces of Westerly Granite and Carrara Marble samples.When the Sommerfeld number 30 to describe friction forces as a function of slip-rate in the three lubrication regimes  in presence of viscous fluids. In this model, the behaviour of the asperities and/or the fluid during shearing is assimilated to the behaviour of some bristles whose deflection can be described asz is the deflection of the bristles, v is the slip rate and at steady state zss = g(v)sgn(z). The friction generated from the bending of the bristles isThe LuGre dynamic friction model was proposed by Canudas de Wit et al.σ0 is the stiffness, σ1 a damping coefficient and σ2 a viscous coefficient.where g(v) seizes Coulomb friction and the Stribeck effect and it can be written as Fs corresponds to the stiction force , and Fc is the Coulomb friction force, i.e., the force necessary to continue sliding in absence of lubricants, v is the relative slip-rate between the moving solid bodies. The characteristic slip-rate of the Stribeck function vs determines how quickly g(v) approaches Fc and depends on fluid viscosity and loading conditions. As suggested by Bo and Pavalescu45, the α parameter ranges between 0.5 and 1. Instead, f(v) is the viscous friction46 and typically is given in the form f(v) = σ2 · v47. In this work, we found According to the model, in mixed and hydrodynamic regimes, the friction force for constant slip-rate at steady state is given by:μdyn, defined as the shear stress divided by the effective normal stress σeff . We assumed that all the mechanical energy is converted into heat and no heat is lost by radiation, so the heat flux Q = 0.5 · τ(t)·V is function of time t and the the radial distance r from the centre of the sample. A Neumann boundary condition was applied to the bottom external edge of the model . At the inner boundary between the slip zone and the wall-rock, the continuity of the solution was granted. The thermal properties of the slip zone were defined as a linear combination of the thermal properties of the fluid and of the rock which are reported in Supplementary Table Keff = (1 − ϕ)·Kr + ϕ · Kf and (ρ·C)eff = ((1 − ϕ)·ρr·Cr) + (ϕ·ρf·Cf) where r and f are related to the rock and fluid properties, respectively.The model reproduced the sizes of the experimental sample,  with 1464 triangular mesh elements. Two different materials were used to simulate the slip zone and the bulk material. The slip zone was represented as a 13 μm-thick highly porous media. In agreement with the roughness measurements reported in Supplementary Figure i identified the two materials. The temperature in the slip zone was taken when the μdyn was reached  can be written as:dU = CΔτ/μ′dL is the slip increment, μ′ shear stiffness, Δτ the stress drop and Gc the fracture energy.The energy change Therefore we may write the energy delivery rate in a simplified form:C a geometrical dimensionless constant.with For the crack to propagate the requirement is that L with U by using L = μ′/(CΔτ)U we obtain 1/2Δτ U ≥ C Gc where the stress drop is Δτ = τ0−τdyn. Therefore we obtain:substituting Supplementary InformationPeer Review File


Pilocarpine is an M3 muscarinic agonist that is widely used for the treatment of xerostomia caused by various diseases and medical conditions. Pilocarpine induced the secretion of salivary fluid in perfused submandibular glands of rats. The secretion of salivary fluid observed after removal of pilocarpine was referred to as residual fluid secretion. The volume of fluid and time of the residual secretion depended on the dose of pilocarpine. Such a residual effect of pilocarpine was observed on fluid secretion via the paracellular pathway and oxygen consumption. When a muscarinic antagonist was added to the perfusate immediately after cessation of pilocarpine, residual secretion of salivary fluid did not occur. These observations indicate that the residual secretion of salivary fluid is a characteristics of the interaction of pilocarpine with muscarinic receptors. Salivary secretion is controlled by the autonomic nervous system, and occurs in response to stimulation by neurotransmitters released from nerve endings , 2. SimuSalivary hypofunction results in xerostomia (dry mouth), which is caused by numerous medical conditions including in patients who receive therapeutic irradiation to treat head and neck cancer, autoimmune diseases such as Sjögren’s syndrome, graft-versus-host disease and as side effect of using certain medications , 7. LongPilocarpus jaborandi, is a muscarinic agonist. It binds to M3 muscarinic receptors in salivary glands and increases the salivary secretion for an adequate length of time; in addition, pilocarpine can be administered by mouth and has tolerable side effects -[3H]scopolamine methyl chloride competition binding assay, and the association and dissociation rates of pilocarpine were higher and lower than that of carbachol, respectively [In Chinese hamster ovary cells expressing the M3 muscarinic receptors, the association and dissociation rate constants of the muscarinic agonists were measured using a ectively . Furtherectively . Furtherectively , 39. TheIn this study, we demonstrated that pilocarpine induced the residual secretion of salivary fluid in perfused submandibular glands of rats. Such an effect of pilocarpine was observed in the submandibular glands of mice . HoweverIn conclusion, pilocarpine provoked residual secretion of salivary fluid in perfused submandibular glands of rats. The effect of pilocarpine is caused by the residency of pilocarpine on muscarinic receptors in the submandibular gland.S1 FigAfter perfusion with normal or pilocarpine-containing perfusate, the submandibular glands were immediately fixed in neutral phosphate buffered 3.7% formalin. The tissue sections from the formalin-fixed tissues were stained with hematoxylin and eosin for histology observation. (a) Control and (b) 1 mM of pilocarpine-perfused rat submandibular glands. Scale bar = 20 μm.(TIF)Click here for additional data file.