VS profiles were used to map both shallow structural profiles and groundwater depth, thus demonstrating that basin-scale variations in hydrological state could be resolved using this technique. The same array was utilized for detection of regional and teleseismic earthquakes and evaluated for long period response using records from the M8.1 Chiapas, Mexico 2017, Sep 8th event. The combination of these two sets of observations conclusively demonstrates that regionally extensive fiber-optic networks can effectively be utilized for a host of geoscience observation tasks at a combination of scale and resolution previously inaccessible.We present one of the first case studies demonstrating the use of distributed acoustic sensing deployed on regional unlit fiber-optic telecommunication infrastructure (dark fiber) for broadband seismic monitoring of both near-surface soil properties and earthquake seismology. We recorded 7 months of passive seismic data on a 27 km section of dark fiber stretching from West Sacramento, CA to Woodland, CA, densely sampled at 2 m spacing. This dataset was processed to extract surface wave velocity information using ambient noise interferometry techniques; the resulting  InSAR3), or low-resolution volumetric datasets (e.g. GRACE5) which require assimilation with point measurements to provide finely resolved operational parameters6. Remote sensing measurements often also suffer from temporal undersampling due to satellite pass frequency. More recently, seismic ambient noise interferometry7 has been leveraged to provide broader information on ground water storage; unfortunately limited permanent seismic networks present a challenge for these approaches.Hydrogeologic and seismological data collection are two domains for which the absence of high spatio-temporal resolution data is particularly acute, with significant impacts on our ability to characterize near-surface soil properties, groundwater systems, and seismic events. Even relatively basic subsurface hydrological parameters such as water table depths in surficial aquifers suffer from severe undersampling in both space and time. While heavily monitored basins often have a multitude of wells providing subsurface access, they are neither uniformly distributed nor frequently monitored resulting in heterogeneous datasets requiring manual quality control, curation, and analysis. The few basin-wide hydrogeological data sources, typically based on satellite remote sensing technologies, provide only surficial property estimates like soil moisture8.Likewise, seismological data collected using existing permanent networks often have spatial regions which suffer from significant spatial undersampling, particularly in areas distant from major plate boundaries, resulting in challenges when attempting to detect and locate small natural and induced events. The case of small magnitude induced events is particularly problematic since the basins where oil and gas production, wastewater injection, and carbon dioxide sequestration occur are often distant from historically seismogenic faults and the associated permanent seismic networks. Network sparsity increases the minimum event size for detection, results in statistical biases in the catalog, and greatly increases depth uncertainty for local events. Recent studies focusing on seismic catalog completeness in California have determined that even M2 events cannot be detected in the majority of the Sacramento and San Joaquin Basins using the existing network stations10; other examples include using social media proxies as sensors11 or MEMS accelerometers in pervasive stationary devices such as personal computers12. Broader efforts to leverage networking and sensor technologies related to the Internet-of-Things (IoT) for seismology are developing but still in their infancy13.These spatio-temporal undersampling problems, for both hydrological and seismological measurements, can be remedied by re-purposing ubiquitous sensing platforms already deployed at scale. A recent example of such an approach is the utilization of smartphone accelerometers to measure strong ground motion as part of earthquake early warning applications as shown in previous studies14, environments rich in ambient seismic noise. Given the ubiquitous nature of installed telecom fibers, few studies have explored use of this resource for sensing applications. An early experiment explored the use of Brillouin Optical Time Domain Analysis (BOTDA) to monitor temperature over previously installed telecom fiber15; however, these studies were conducted primarily to provide network integrity information rather than for environmental sensing. In a seismological context, several recent studies18 have demonstrated the benefits of leveraging urban telecom infrastructure at a small scale. Most recently, a study in southwest Iceland19 provided an excellent example of utilizing telecom fiber for detecting local earthquakes and measuring co-seismic strain measurement over a short transect.An alternative approach is to exploit components of the built environment to serve as distributed sensor networks. In this case we couple the use of unlit subsurface fiber-optic cables, commonly referred to as “dark fiber” since they are not utilized for data transmission, and distributed acoustic sensing (DAS) to provide such a spatially extensive sensing platform. The vast majority of fiber-optic cables in the earth’s near-surface were installed exclusively for the purpose of telecommunications. Due to high cost of fiber-optic installation, typical commercial practice is to deploy significantly more capacity, as measured by fiber count, than required; this practice, combined with advances in bandwidth available per fiber, have yielded a surplus of available fibers that remain unused. The US footprint of such unused fiber networks is massive with tens of thousands of linear kilometers of long distance fiber-optic cables available for lease or purchase in the current environment. One notable aspect of such dark fiber network components is that they tend to utilize existing “right-of-way” corridors along roads and rail connections22. The technique exploits changes in Rayleigh scattering induced by extensional strain; these measurements have now been quantitatively compared to point seismic recordings at both intermediate23 and low frequencies24 and utilized for a host of tasks including vertical seismic profiling25, near-surface soil property estimation30, surface refraction tomography31 and earthquake seismology32. DAS has created a recent paradigm shift in applied geophysics by enabling seismic measurements at a combination of high frequency (kHz range), large distances (tens of km), and fine spatial sampling , a combination previously unavailable with conventional sensors at moderate costs. We should note that DAS is distinct from long-range optical interferometry approaches which provide even greater measurement distances but sacrifice spatial localization; this class of techniques was recently demonstrated33 as an approach for seismic detection utilizing trans-oceanic cables. While prior studies have convincingly demonstrated the value of dense networks for seismic imaging as well as a range of other purposes34, the high costs associated with massive nodal deployments over long time periods has precluded their use in many contexts.Distributed Acoustic Sensing (DAS) is a recently developed technique which utilizes coherent optical time-domain reflectometry to accurately measure the phase and amplitude of vibrations along an optical fiberIn this study, we demonstrate the application of DAS utilizing dark fiber for measurement of seismic wavefields at the sub-basin scale with an extremely fine spatial sampling (2 m) over long time periods; the resulting ultra-dense dataset is utilized for both hydrogeological/near-surface characterization, using ambient noise interferometry, and the detection of seismic events, both regional and global. This combination is perhaps a new frontier which leverages investment in built infrastructure to greatly extend the reach and sampling of existing permanent monitoring networks.Our study utilized dark fiber components of ESnet’s Dark Fiber Testbed. ESnet, a US Department of Energy (DOE) user facility, provides high-performance unclassified network infrastructure to connect DOE research sites including high performance computing (HPC) facilities and data-intensive instrumentation e.g. x-ray, neutron, and nanoscience facilities. The Dark Fiber Testbed is a 20,920 km  network of short and long haul telecommunication fiber designed for testing novel network communication equipment and protocols. The network consists of single mode telecommunication fibers of varying age and installation technologies and hence is an excellent proxy for existing commercial network components. This study is one of the first experiments that utilizes this massive network for sensing purposes. Figure 35 have mapped the surface sediments as a mixture of poorly sorted Holocene alluvium near the Sacramento and finer-grained Holocene basin deposits deeper in the flood plain. Partially lithified sediments from the Tehama formation have been mapped from approximately 50 m to greater depths36. The segment of dark fiber we utilized for this study, shown in Fig. The site of our study was a transect located in the Sacramento River flood plain, north and west of Sacramento, CA. The geology of the site consists largely of Quaternary sediments including a sequence of silts and clays underlain by fine sands. Prior regional studiesThe seismic dataset presented in this study was recorded between July 28th, 2017 and Jan. 18th, 2018. The DAS interrogation unit  was installed in a telecommunication Point-of-Presence (PoP) facility in West Sacramento. Hardware details on the installation are described in Methods Section 0.2. Ambient seismic noise was recorded using the DAS IU at 500 Hz sampling with a spatial sampling of 2 m; the gauge length was 10 m and fixed in hardware. Data was streamed continuously to large capacity (8 TB) USB-3 external hard drives that were exchanged on a weekly basis.While the surface geometry of the dark fiber network was known before deployment, the mapping to linear fiber location was established by sequential impact tests at surface locations surveyed with high accuracy differential GPS. This is necessary due to the common practice of including spools of slack cable during telecom installation, an approach that makes the mapping of surface geometry to linear fiber location more complicated. Impact locations were observed on individual DAS gathers in terms of fiber distance, coordinated by GPS time; by establishing true DGPS coordinates for these locations, we were able to compensate for slack effects when mapping back to the previously surveyed deployment geometry. The resulting geometry likely has an uncertainty on the order of 5 m due to the interpolation process along the transect.After the network geometry was established, DAS signal strength, amplitude, and periodicity were examined to evaluate noise characteristics along the array. Dominant noise features include several regional highways, diffuse urban noise, and energy from local railroad activity. Qualitatively, the highest quality data was observed on a straight section of fiber starting beyond West Sacramento and extending to the noise field of Interstate 5, shown as the highlighted red profile in Fig. 7. Ambient noise interferometry is an established group of seismological techniques that utilize environmental vibrations from either near-surface or subsurface sources to retrieve coherent seismic information, referred to as empirical Green’s functions41. More recently, several authors28 have demonstrated that such techniques could be utilized to transform infrastructure noise in the 2–30 Hz band  into accurate and stable 1-D estimates of shear wave velocity from 0–30 m depth using DAS. While prior studies have examined data acquired on a fit-for-purpose array, the processing strategy proposed can be easily adapted to dark fiber deployments.Prior studies have demonstrated the potential of utilizing ambient noise interferometry and classical sensors to detect fluctuation in groundwater state45 to infer shear-wave velocity (VS) profiles with a multimodal inversion strategy. Figure 46 which utilizes a novel multimodal objective function47 which does not require mode numbering. Supplementary Fig. The present study images near-surface shear wave structure using infrastructure noise generated by freight trains operating along a 6600 m subsection of the dark fiber array  as shown in Fig. m, hence the measured water table matches within error.Comparisons to our limited ground truth database reveal that the interfaces of the VS30, the travel-time average of Vs over the top 30 meters, calculated directly from the surface wave inversion results. VS30 is a widely used indicator of seismic site conditions and is readily obtainable from surface wave inversion48; both the values and the lateral variations of VS30 are useful data products that the dark fiber array can provide and can be compared to local estimates made as part of geotechnical studies.Also shown in Fig. Vs identified as the water table to direct point measurements in the one well with sufficient temporal sampling information located on our transect. The well (W1) is located at the southeastern end of the high S/N section of dark fiber near the intersection of County Road 126 and the Old River Road, shown as a red dot on Fig. Vs increase (3.8–4.6 m below the surface) correlates to the measured surficial water table depth. We should note that an increase in Vs at the water table is not predicted by classical rock physics models which assume that shear modulus is not sensitive to saturation; despite this, past field studies49 have provided detailed observations of such sensitivity, presumably due to the impact of moisture on soil cohesion for dry surficial materials.To evaluate the utility of the DAS ambient noise inversion results for hydrogeological monitoring, we compare the transition in Vs, the dark fiber array also allows for time-lapse monitoring of the subsurface, enabling measurement of hydrologic transients in the near-surface. Traditionally, because sensors are rarely dense enough for imaging, time-lapse monitoring results are typically presented as an apparent-velocity perturbation along the path connecting two sensors, without identifying where on this path the changes are occurring. This level of detail is insufficient for proactive management of groundwater resources, given that both vertical and lateral changes of the groundwater level could affect the overall sustainability of the aquifer. With the dark fiber profile enabling time-lapse imaging of the near surface, changes in groundwater levels could be resolved with spatial-temporal resolutions pertinent to groundwater management.As mentioned previously, the Sacramento Basin is a dynamic hydrologic environment with multiple productive aquifers, active groundwater production, irrigation, and river interactions. Besides static profiles quantifying Figure 51, in part because broadband seismic stations are sited in hard-rock locations where background noise is low53. Areas of less-competent geology, like sedimentary basins, therefore correlate with poor catalog completeness; the magnitude of completeness is Mc 2–3 in the Sacramento and Southern San Joaquin Basins compared to Mc 0.5–2.4 in the San Francisco Bay Area or Mc 0.5–1.8 in Southern California55. Thus, despite the greater Sacramento area hosting significant gas production, underground gas storage, and high-volume waste water disposal, all of which can impact seismicity, the Sacramento Dark Fiber DAS array is located 30 km away from the nearest networked short-period seismometer (NDH) and 62 km away from the nearest broadband seismometer (AFD).Seismic network detection thresholds are highly heterogeneous, even across regions known for dense seismic monitoring like the western United States and Japan19, fiber installation in a standard plastic conduit does not preclude sufficient sensor coupling required for the detection of earthquakes, but the case of recording DAS data with repurposed telecommunications fiber is yet untested at regional scales. To explore this question with the Sacramento Dark Fiber DAS experiment we extract raw strain-rate waveforms for major global and regional earthquakes that occurred during the continuous recording interval , for example, may be dominated by thermal expansion of the fiber-optic cable (strains on the order of 1 × 10−6) depending on the frequency studied as well as the depth, composition, and condition of the fiber-optic cable and conduit. Recent studies56 have used shallow hydrogeologic pump tests in a well with a fiber-optic cable to show that DAS has sensitivity to 9.4 × 10−3 Hz (period = 1080 seconds) oscillations in strain induced by the variable confining pressure, presumably due to Poisson effects. This subject is complicated by the known directionality of DAS cables58, which for the horizontal geometry of telecommunications dark fiber cables is theoretically insensitive to vertically-incident compressional motion (P-waves).While long period sensitivity is a major limitation of many inertial seismic sensors , the long period response of DAS is currently a topic of active research with only limited available dataTo explore the long period sensitivity of the Dark Fiber DAS array to teleseismic events, we extract raw strain-rate seismograms from the largest earthquake recorded during the experiment, the M8.1 2017-Sep-08 Chiapas, Mexico earthquake Fig. . We obseM ≈ 4) generate ground motions on the Sacramento array that have equal or lesser amplitude than local moving vehicles, however anthropogenic seismic signals typically are dominant in a higher frequency band (5–30 Hz). Figure Telecommunications cables are commonly routed along railways, roads, and through high noise urban areas. We find that major regional earthquakes  has a significant effect on DAS recorded ground motion see Fig. . The fibμm single-mode fiber from one of these cables. In a few locations, trenching was not possible so directional boring was used to install a large casing conduit , inside of which the 12 smaller conduits were pulled. Depth of boring varied between one meter and a few meters when navigating around various culverts, sections of road and railway, and other obstacles. In some instances the casing was not required, or a steel casing may have been used. A third mode of installation used for approximately 300 m of the dark fiber array involved attaching a 20–25 cm diameter steel casing directly to the elevated rail line where it crosses a section of protected wetlands, the Sacramento Bypass Wildlife Area. Inside this attached conduit, the 12 HDPE conduits were installed as the boring method described above.Installation information for the Sacramento Dark Fiber DAS array provides clues as to the heterogeneity of fiber-soil coupling across our experimental profile. Cable installation occurred in 1999–2000. Most of the fiber was pulled through one of 12 high-density polyethelene (HDPE) conduits  that were buried together in a trench at 1–1.5 m and backfilled with soil before installing the fiber cable inside. Each fiber cable contains 84 gel-filled, loose-tube Corning LEAF fibers that are polythelene jacketed and steel-armored. The DAS data were recorded using a single 9/125 Figure 38, a variety of recent projects have utilized ambient noise approaches to probe hydrologic cycles61 and aquifer structure62 although typically using a sparse network of stations. Such studies have typically relied on the microseisms band as a noise source (0.1–1 Hz) and hence are observing averaged velocity perturbations over significant vertical extent, often to km depths. Despite this, such approaches have yielded convincing correlations with environmental parameters such as groundwater level59 although often for larger perturbations (10 s of m perturbations) in comparison to our study. In contrast, our experiment is densely sampled in space and utilizes infrastructure noise in the 0.5–18 Hz range; this enables adoption of surface wave inversion approaches utilized in the geotechnical community63 and the monitoring of very shallow features with some degree of depth resolution. Unfortunately, the minimal precipitation and small perturbations (1 m) in groundwater level which occurred during our study period precluded effective observation of hydrologic variations. Despite this fact, the static structure observed was consistent with our available ground truth dataset.While the focus of our study was the specific utilization of installed telecom fiber probed by DAS for seismic sensing, our static imaging and monitoring results are consistent with and rely on broad advances in the field of ambient noise seismology applied to the near-surface. Beyond the foundational studies cited previously64 which utilized a more classical sensor array (4.5 Hz geophones) confirms the utility of rail noise as an imaging source for both P as well as S-wave imaging. These observations bode well for future dark fiber exploitation; since many telecom fiber installations use railway right-of-ways for installation, judicious selection of fiber paths can exploit this powerful source of seismic energy. Our experiment also provide an unusual case study of large (12k+ channel) array recording with dense receiver spacing (2 m). The only comparably sized experiments (5000+ sensors) are massive nodal deployments, which have recently been leveraged for similar event detection65 and ambient noise67 applications.As mentioned previously, the ambient noise aspect of our study was also greatly enhanced by the broadband seismic signal generated by rail traffic co-linear to our measurement fiber. Recent seismic interferometry examples from the 2014 Belen experiment68 reported an average Vs30 of 234 m/s which is broadly consistent with our surface wave inversion values of (210–280 m/s), a NEHRP class D soil environment.Outside of the validation datasets presented previously, prior studies in close proximity to our site are not extensive. Geotechnical evaluations, conducted in support of levee evaluation and construction broadly agree with our conclusions. At a location near our fiber profile, a past study evaluating levee safety in West SacramentoAs demonstrated in the prior sections, DAS-based seismic measurements acquired using dark fiber can provide a wealth of information relevant to near-surface seismic property estimation, hydrologic state, and natural seismicity. Measurements using dark fiber also have advantages in a host of situations ranging from marine to urban scenarios where classical seismic networks are challenging to execute.7, the spatial resolution of such investigation is typically on the order of km due to sparse sensor distribution. Recent advances in large N processing approaches32 also offer strategies for leveraging dense arrays for detecting small seismic events.An obvious strength of dark fiber DAS deployments, demonstrated in this study, is the potential to record data across long (10 s of km) transects at high spatial resolution without any required sensor installation or power source. Chains of such deployments could be utilized to provide true basin scale sensing; the Sacramento Basin’s central width (120 km) could be spanned using only 4 independent interrogation units and existing dark fiber resources, providing an unprecedented sensing resource. While basin scale hydrogeophysical monitoring studies using point sensors and ambient noise have been recently conductedA second advantage of utilizing dark fiber for seismic measurements is dense non-invasive coverage in urban areas where diverse deployment and permitting environments challenge classical acquisition strategies. The present study provides coverage spanning urban (Sacramento), suburban, and rural zones without the typical landowner permission effort, permitting, and survey work required for deployment in occupied areas.While not demonstrated in this study, dark fiber can also be utilized for observations in the transition zone and offshore domains, areas where almost no measurements exist at present due to the high cost of tethered marine observatories including seismometers. Offshore DAS measurements would be particularly useful for improving the hypocenter accuracy for small events occurring on marine faults and earthquake early warning in subduction zones. Limitations of using DAS and dark fiber for offshore observations include (a) distance constraints for DAS measurements on single mode fiber for existing optical chains, currently in the range of 30–40 km, (b) the considerably higher cost for dedicated use of fibers in transoceanic cables, (c) the lower density of offshore cable routes which reduces coverage. Having said this, offshore dark fiber recording provides a clear future opportunity to extend the domain of seismological measurements into previously uninstrumented regions.70 provide a path to potentially handle the much larger datasets generated by dark fiber studies.Despite these opportunities, challenges exist to fully exploit these conceptually novel sensing networks. Extremely large data volumes are among the most pressing, although solvable, problems; at maximum acquisition rates, a single interrogator can generate upwards of 20 TB/day. Combining these large N deployments with the long time periods required for ambient noise processing and monitoring yields raw data volumes that exceed the capacity of the computational infrastructure available to most researchers. The array in this study, which included 12000 channels sampled at 500 Hz, generated 128 TB of raw data in the first 3 months of operation and approximately 0.3 PB of raw data when the system was demobilized. Volumes of this size require careful consideration of data transport requirements, storage, archiving, and automated processing to be successfully utilized. Fortunately, on-going efforts to solve I/O and computational barriers in ambient noise studiesThe Silixa iDAS unit utilized in this study was installed on a vibration isolated table located in the West Sacramento PoP. Vibration isolation consisted of a 18 × 24′′ Nexus Breadboard (Thorlabs) with passive Sorbothane feet (Thorlabs) placed on a durometer 70 Sorbothane sheet. This assembly was placed on a rack shelf within the PoP cage where the utilized dark fiber was terminated in a standard fiber-optic patch panel with SC-UPC connection. Connection to the Silixa iDAS was made using an SC-UPC/SC-APC single-mode patch cable. An optical time-domain reflectometer (OTDR) was used to evaluate fiber integrity prior to recording. An OTDR trace measured a total loss of 20.8 dB over the full fiber length of 101 km at 1550 nm, or an average loss of 0.2059 dB/km.https://www.globus.org/, last accessed: 2018-05-21) from a networked data transfer node. The storage server was linked to five RAID6 disk arrays and the full dataset was striped to improve performance. Primary processing was carried out on a 32-core GPU server connected to the storage server via a fast GB switch. Final results were visualized using a combination of MATLAB (Mathworks) and the ObsPy package.As mentioned in the main manuscript, the data was collected at 500 Hz in the iDAS native format in the form of raw 1 minute records at 2 m spatial sampling. Files were written to a local USB3-connected 8 or 16 TB external hard drive. To maintain continuity of the dataset, hard drives were replaced on a weekly or biweekly basis and manually transferred from West Sacramento CA to Berkeley CA, where the data were uploaded to a local RAID storage server using the Globus protocol  so each epoch utilized 40 minutes of train noise for analysis, independent of the number of days required to accumulate this stack. These N = 40 stacks were typically generated over approximately 10 days.ν = 0.5) was used and a stack count of 40 was sufficient for reaching temporal stability. Prior studies71 document the utility of phase weighted stacks in ambient noise analysis. A slant stack was then applied to the stacked common virtual-shot gathers to transform the data from space-time domain to frequency-velocity (dispersion) domain.After selection and to prepare the raw records for noise correlation, static offsets and linear trends were removed, followed by temporal decimation down to a coarser sampling rate of 8 ms. Next, a temporal normalization with running-absolute-mean was applied over a 0.5-second running window. The frequency content of the data between 0.5 Hz and 18 Hz was then balanced with a spectral whitening step. Finally, in each of the 1-minute records, the noise of the virtual-source channel was cross-correlated with the rest of the channels’ records to form a common virtual-source gather. To achieve good SNR with a minimal stack count, phase-weighted stacking  the objective function can be very efficiently evaluated without root finding, thus allowing global search, and (b) non-labeled multimodal dispersion data can be utilized as an input.As discussed in prior work46 as part of search and model selection. We adopted a sparse parameterization for the search problem assuming a four layer model for each array subsection and solved for Vs and layer thickness for each location. For each 1D inversion, a Monte Carlo pool size of 1 × 106 models was used. Search bounds of the model parameters are shown in Table Vs with depth, likely a valid assumption in this geology. As can be seen in Table Because of the nonlinear nature of the problem and the low computational costs of the Haskell-Thomson determinant method, Monte Carlo sampling was usedWe identified earthquake records in the continuous raw DAS dataset using catalogued origin times and approximate travel times to Sacramento for the 1-D iasp91 Earth velocity model. 1-minute duration earthquake records were merged together, and then, if desired, stacked with a mean average of traces over a specified length (100 m = 50 traces averaged and plotted at the midpoint), prior to the application of a specified bandpass filter to remove unwanted or uninteresting signals. In Fig. Data on the reference groundwater monitoring well discussed (W1) was acquired from the CASGEM database. As mentioned in the text, the well is referred to as the Sac Bypass Shallow Well (State well ID 09N04E20-N001M and CASGEM ID 25619). The well is located in Yolo County at 38.6062 N, −121.5602 W with a surface elevation of 6.55 m (21.49 ft) above sea level; the well is associated with the Yolo County Water Resources Association (WRA) with measurements conducted by the CA Department of Water Resources (DWR). The well is completed with slotted PVC and a sandpack over a 6.1 m (20 ft) interval from 24.4 to 30.5 m below ground surface. Water table depths were measured manually at an irregular intervals (every 1 to 3 months) with a reported accuracy of 0.3 cm (0.01 ft).Supplementary Section


Giant cell tumor is known to be a benign neoplasm that arises most commonly in the long bones, while cases in the spine are rare. Recently, denosumab, a monoclonal antibody that inhibits receptor activator of nuclear factor-kappa β ligand, has been used to treat patients with giant cell tumor. However, there are few reports of total en bloc spondylectomy being used for paravertebral giant cell tumor lesions following denosumab therapy.Our patient was a 20-year-old Japanese woman with a 4-month history of lower back pain. A spinal computed tomography scan and magnetic resonance imaging of her lumbar spine revealed an osteolytic lesion involving the L3 vertebral body, and the tumor extended toward the left side of the paravertebral soft tissue and into the left pedicle. The lesion was diagnosed as a giant cell tumor by needle biopsy. Denosumab treatment calcified the paravertebral giant cell tumor lesion and the tumor vertebral body was removed completely by total en bloc spondylectomy.This case report describes a patient with a paravertebral giant cell tumor who was successfully treated by preoperative denosumab injection followed by total en bloc spondylectomy. Giant cell tumors (GCTs) account for 4–8% of all primary bone tumors, and they are most frequently located in the juxta-articular metaphysis of the long bones , while 2A 20-year-old Japanese woman with a 4-month history of severe lower back pain was referred to our out-patient department. She had no history of fever, trauma, weight loss, or previous infection.Radiographic analysis showed collapse of the left side of the L3 vertebral body and swelling of the iliopsoas muscle. Spinal computed tomography (CT) revealed an osteolytic lesion involving the L3 vertebral body and surrounding soft tissue, causing vertebral body collapse Fig. a, b. MagFollowing denosumab treatment, she underwent two-stage (anteroposterior) L3 TES. Stage 1 utilized a posterior approach for resecting the posterior vertebral component; the total operation took 5 hours 16 minutes and the total bleeding was 1520 ml. Stage 2 utilized an anterior retroperitoneal approach for resecting the anterior vertebral component followed by intervertebral cage insertion; the total operation took 6 hours 43 minutes, and the total bleeding was 2320 ml  [Denosumab was recently approved for GCT, and for those with severe osteoporosis . Denosumse (CKD) . To prevet al. reported two cases of sarcoma that arose from bone GCTs by long-term denosumab administration [The need for postoperative denosumab is still to be determined. Inhibition of RANKL may increase the risk of new malignancies by inducing immunosuppression . Broehm stration . On the stration , there hIn conclusion, this case report describes a patient with paravertebral GCT who was successfully treated by preoperative denosumab injection followed by TES.


Traumatic brain injury remains a significant cause of death and disability in the USA. Currently, there are no effective therapies to mitigate disability except for surgical interventions necessitating a need for continued research into uncovering novel therapeutic targets. In a recent study, we used a rodent model of penetrating traumatic brain injury known as penetrating ballistic-like brain injury (PBBI) to examine the role of innate immunity in post-traumatic secondary injury mechanisms. We previously reported that the inflammasome, a multiprotein complex composed of apoptosis-associated speck-like protein containing card and caspase-1, plays a role in secondary cell death mechanisms after PBBI, including inflammatory cell death (pyroptosis).In the current study, we used flow cytometry analysis to evaluate activated microglia and CD11b-positive leukocytes after PBBI and assessed inflammasome activation and pyroptosis of specific cellular populations. Sprague-Dawley male rats underwent PBBI or sham-operated procedures and ipsilateral cortical regions processed for flow cytometry and cellular analysis. Flow cytometry results were compared using one-way ANOVA followed by Tukey’s multiple comparisons.At 48 h following PBBI, there was an increase in activated microglia and infiltrating leukocytes compared to sham controls that were associated with increased caspase-1 activity. Using a florescent probe to identify caspase-1 activity and a fluorescent assay to determine cell viability, evidence for pyroptosis in CD11b+ cells was also determined. Finally, while post-traumatic treatment with an anti-ASC antibody had no effect on the number of activated microglia and infiltrating leukocytes, antibody treatment decreased caspase-1 activity in both resident microglia and infiltrating leukocytes and reduced pyroptotic CD11b+ cell death.These results provide evidence for inflammasome activation in microglia and infiltrating leukocytes after penetrating traumatic brain injury and a role for pyroptotic cell death in the pathophysiology. In addition to inhibiting neuronal cell death, therapeutic treatments targeting inflammasome activation may also provide beneficial effects by reducing the potentially detrimental consequences of activated microglia and infiltrating CD11b+ leukocytes following penetrating traumatic brain injury. Penetrating traumatic brain injury (PTBI) includes all traumatic brain injuries that are not the result of a blunt mechanism –3. PTBI As part of the pyroptotic process, damaged neural cells form specks that are composed of apoptosis speck-like staining protein containing a caspase recruitment domain (ASC) and caspase-1 , 23. ASCMicroglia play an important role in the immune system of the central nervous system (CNS) by acting as antigen presenting cells and surveying tissue for immunogens –34. ImpoIn the present study, we sought to extend our previous investigations using the PBBI model  to clariAnimal procedures were approved by the University of Miami’s Institution of Animal Care and Use Committee and adhered to the ARRIVE guidelines and those established by the National Institute of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats 280–350 g) aged 3–5 months were used for all experiments. Male rats were used in this initial study because males are at higher risk of traumatic brain injury (TBI) than females, with the highest male-to-female ratio occurring in adolescence and young adulthood . However g aged 3Rats underwent PBBI or sham procedures as previously described . Rats welow “resting” microglia , CD45intermediate-activated microglia , and CD45high-infiltrating myeloid-lineage cells . Activated microglia increase their expression of CD45 compared to steady state surveying microglia while infiltrating leukocytes, including macrophages, monocytes, and neutrophils, express the highest amounts of CD45 [At 48 h post-surgery, all rats were anesthetized with a high dose of 2–5% isoflurane for 5 min and perfused transcardially with ice-cold PBS for 6 min. The ipsilateral cerebral cortex was dissected on ice and placed in ice-cold Hank’s Balanced Salt Solution. The methods used for flow cytometry have been recently described for cerebral cortical and hippocampal analysis , 55. Bri of CD45 , 57. Celn = 5 per group). Samples were coded prior to flow cytometry, and the codes were broken after analysis with Kaluza and prior to statistical analysis. Statistical analyses were performed using Prism 6 . All measures were expressed as mean ± standard error of the mean with p ≤ 0.05 considered significant in all statistical tests. Flow cytometry results were compared using one-way ANOVA followed by Tukey’s multiple comparisons.Rats were randomly assigned to each experimental group including sham operated, PBBI + PBS, and PBBI + anti-ASC , as evidenced as tight cell clusters on the graph with very few cells in the CD45int and CD45high range , activated microglia , and infiltrating CD11b+ leukocytes . Although, there were significantly less surveying microglia in the vehicle and treated animals 48 h after PBBI compared to sham animals. However, there was no significant difference between vehicle and treated groups. The CD45 and CD11b expression pattern of surveying microglia  in vehicle and treated cortices differed from the expression pattern of surveying microglia  in sham cortices showing that the cells were less tightly clustered indicating that the microglia increased their expression of both CD45 and CD11b after injury when compared to sham. Conversely, there were significantly more activated microglia  in vehicle and treated animals 48 h post-PBBI compared to sham animals. Treatment with anti-ASC did not significantly change the number of activated microglia when compared to vehicle. Compared to sham, the number of infiltrating CD11b+ leukocytes  also significantly increased in vehicle and treated animals 48 h after injury. Similar to the number of activated microglia , the number of infiltrating leukocytes  did not significantly change after treatment with an inhibitor of ASC. These results indicate that PBBI increases the number of activated microglia , recapitulating stereology results from a previous study [high, CD11b+). However, anti-ASC treatment did not alter the number of either type of cell.Following PBBI, cells in vehicle and anti-ASC treated cortices were gated into three populations: surveying microglia  and infiltrating CD11b+ leukocytes  in vehicle animals 48 h after PBBI  and the infiltrating leukocytes population . These results suggest that PBBI increases caspase-1 activity in activated microglia and infiltrating CD11b+ leukocytes and that inhibition of ASC decreases caspase-1 activity in these cells. Therefore, inflammasome protein ASC contributes to inflammasome activation and caspase-1 activity in microglia and infiltrating leukocytes in PBBI.To determine the role of ASC on caspase-1 activity in microglia and infiltrating leukocytes in PBBI, PBBI animals were treated with anti-ASC, a biological inhibitor of adaptor protein ASC, and flow cytometry was conducted to measure caspase-1 activity before and after treatment. A FAM-FLICA florescent probe, which only binds to cleaved and activated caspase-1, was used to measure caspase-1 activity in cells isolated from the cortices. Using the established gates for CD45 and CD11b, caspase-1 activity significantly increased in the activated microglia , live CD11b+ cells that express caspase-1 activity , necrotic CD11b+ cells , and pyroptotic CD11b+ cells   was used to determine cell viability . The LIVgh) Fig.  top row.gh) Fig. , and thahigh, LIVE/DEADhigh) and caspase-1 activity live cell  gate, the CD11b+ cells were further gated into “resting” microglia , activated microglia , and infiltrating leukocytes   and activated microglia gate   versus activated population , it was observed that activated microglia were the main source of the live cells with caspase-1 activity and pyroptotic cells in PBBI cortices (Fig. To determine the role of ASC in pyroptosis and caspase-1 activity in microglia and infiltrating CD11b+ leukocytes in PBBI, animals were treated with an inhibitor of ASC and flow cytometry was conducted to determine cell viability and caspase-1 activity. Using the pyroptotic cell gate (FLICAThis study provides several new findings that clarify the underlying pathophysiology of secondary injury mechanisms after severe TBI. We present evidence using flow cytometry density plots that PBBI significantly increases the number of activated microglia and infiltrating CD11+ leukocytes 48 hrs after injury. Likewise, the number of activated microglia and infiltrating CD11+ leukocytes expressing caspase-1 was also significantly increased in cortical inflammatory cells compared to sham-operated animals. Using a FAM-FLICA florescent probe and LIVE/DEAD assay, caspase-1 activity in live and pyroptotic cells was found to be increased in all CD11+ cortical cells after PBBT. Post-traumatic treatment with an antibody inhibiting ASC significantly decreased the amount of caspase-1 activity in both activated microglia and CD11+ leukocytes and reduced the number of CD11+ leukocytes undergoing pyroptosis. Taken together, these new findings indicate that activation of the inflammasome and pyroptosis contributes to the early pro-inflammatory response to severe TBI and supports previous findings showing pyroptosome formation and gasdermin-D expression after PBBT . In addiStreptococcus pneumoniae infection induced pyroptosis in cultured microglia as evidenced by cleavage of caspase-1 and an increase in lactate dehydrogenase release into the culture media [Although previous studies have reported NLRP3 inflammasome activation in microglia and macrophage populations under several experimental conditions , 59–62, re media . Pyroptore media , 64, andre media , 65, 66.+/CD45low, other CNS macrophages and peripheral macrophages exhibit the phenotype CD11+/CD45high. Thus, while both CD11b and CD45 can recognize various subtypes of invading cells including leukocytes and lymphocytes that may participate in the pathophysiology of TBI, the current strategy allowed us to isolate these two major inflammatory cell populations with flow cytometry to evaluate inflammasome signaling.Our flow cytometry experiments revealed an increase in the number of activated microglia 48 h after PBBI that corresponds to increased microglia previously assessed by stereological counts using the same model . These fTo reduce the detrimental consequences of pro-inflammatory processes after TBI, various therapeutic targets and strategies have been investigated with mixed results , 68–70. Based on the current findings and previous studies, it may be hypothesized that in this severe TBI model, inflammatory cell pyroptosis may induce the formation of ASC specks in traumatized cells that are released into the extracellular space leading to maturation of IL-1β , 23. ASCIn summary, our current findings emphasize that a severe model of PBBI induces significant increases in cell-specific inflammasome protein expression which contributes to the inflammatory cell death of microglia and infiltrating leukocytes. Treatment with an ASC neutralization antibody decreased caspase-1 activity and pyroptosis in microglia and infiltrating leukocytes. These data illustrate that resident and invading inflammatory cells actively participate in the early innate immune response to PBBI. The persistence and mobility of these cells may represent important secondary injury processes that aggravate the injury process and potentially lead to the increased vulnerability of more remote brain regions not initially damaged by the primary injury. Continued investigations into the role of abnormal inflammasome activation in specific cell populations will provide critical information regarding novel cellular and molecular therapeutic targets to reduce the acute and more progressive consequences of TBI.


Erythropoiesis is a tightly-regulated and complex process originating in the bone marrow from a multipotent stem cell and terminating in a mature, enucleated erythrocyte.Altered red cell production can result from the direct impairment of medullary erythropoiesis, as seen in the thalassemia syndromes, inherited bone marrow failure as well as in the anemia of chronic disease. Alternatively, in disorders such as sickle cell disease (SCD) as well as enzymopathies and membrane defects, medullary erythropoiesis is not, or only minimally, directly impaired. Despite these differences in pathophysiology, therapies have traditionally been non-specific, limited to symptomatic control of anemia via packed red blood cell (pRBC) transfusion, resulting in iron overload and the eventual need for iron chelation or splenectomy to reduce defective red cell destruction. Likewise, in polycythemia vera overproduction of red cells has historically been dealt with by non-specific myelosuppression or phlebotomy. With a deeper understanding of the molecular mechanisms underlying disease pathophysiology, new therapeutic targets have been identified including induction of fetal hemoglobin, interference with aberrant signaling pathways and gene therapy for definitive cure. This review, utilizing some representative disorders of erythropoiesis, will highlight novel therapeutic modalities currently in development for treatment of red cell disorders. In this review, we hope to provide the reader with an overview of our current understanding of human erythropoiesis, along with a classification of representative disorders leading to a decrease or increase in red cell production with the clinical strategies being used currently or in the near future for management of these patients. The discussion of steady state erythropoiesis provides the framework for understanding these representative disorders and allows the reader to contextualize other diseases, not described herein, within this framework. For a deeper understanding of the molecular bases governing erythropoiesis, the reader is referred to these outstanding reviews published within the last 5 years  to the mature erythrocyte Orkin . The firThe second phase of erythroid maturation involves the differentiation of the nucleated precursors from proerythroblasts to basophilic, polychromatophilic and orthochromatic erythroblasts. This phase is characterized by the gradual accumulation of hemoglobin, progressive decrease in cell size and nuclear condensation ultimately resulting in enucleation  is a humoral cytokine synthesized primarily in the kidney and secreted into the blood stream where it targets erythroid progenitor cells in the bone marrow (Broxmeyer EPO binds to the erythropoietin receptor (EPO-R) which causes receptor homodimerization. Erythropoiesis regulation by EPO is temporally regulated, and human studies have shown that EPO binds to the EPO-R from the CFU-E stage to the polychromatophilic stage  syndromes are presented as an example of anemia characterized by essentially normal medullary erythropoiesis.Hemoglobin synthesis is controlled by two multi-gene clusters located on chromosome 16 (α-like globins) and chromosome 11 (β-like globins). The α gene product combines with the β gene product to form Hb A (α2β2), which is the major form of adult hemoglobin. During fetal life, two γ genes, also located on chromosome 11, combine with α- globin genes to form Hb F (α2 γ 2). A gradual switch from Hb F to Hb A begins before birth and is largely completed by 6 months of age.In β-thalassemia, a mutation in the β-globin gene results in an imbalance between α- and β- globin chains. This results in an accumulation of unstable α-tetramers within erythroid cells leading to premature cell death within the differentiating red cell. This leads to ineffective erythropoiesis with impaired differentiation of maturing erythroblasts at the polychromatic and orthochromatic phase) and the relative proportion of functional α chain synthesis  in adults or Hb Bart’s (γ4) in the fetal period causing hemolytic anemia and ineffective erythropoiesis. Individuals affected by α-thalassemia have variable degrees of anemia, microcytosis and percentage of HbAS from both parents results in the most common and severe form of the disease, Hb SS. However, other compound heterozygous forms of the disease including HbSC, HbS/β0thalassemia and HbS/β+-thalassemia result in sufficient expression of HbS to cause intracellular sickling , stroke, priapism, and acute chest syndrome (ACS). Chronic complications are related to small and large vessel vasculopathy, progressive ischemic organ damage and chronic hemolysis; these include cerebrovascular disease, retinopathy, pulmonary hypertension, gallstones renal failure, hyposplenism, bone disease, hepatopathy and premature death  is a clonal disorder of myeloproliferation in the bone marrow. It is characterized by increased red cell mass associated with the proliferation of the erythroid, megakaryocytic and granulocytic cell lines. It is the most common chronic myeloproliferative neoplasm (MPN) in adults and is virtually always characterized by the JAK2V617 point mutation corresponding to JAK2 exon 14 and less commonly, exon 12. The mutation results in the constitutive activation of the JAK/STAT signaling pathway that is independent of EPO signaling  is the 2nd most common form of anemia worldwide and is associated with significant impairments in quality of life , transforming growth factor-β (TGF-β), tumor necrosis factor (TNF) and interferon-γ (IFN-γ) are found in a multitude of chronic inflammatory conditions and malignancies ,Erythroid lineage and developmental stage- specific expression of transferred globin gene, safe integration and expression of gene with little to no risk of insertional oncogenesis  to reverse transcribe their RNA into complementary DNA (cDNA) which can then be incorporated into the host cell genome for therapeutic delivery of gene elements. Most successful gene therapy trials utilize lentivirus (LV) vectors as they possess the ability to enter into an intact nucleus and integrate into non-dividing cells. This results in high efficiency transduction of genetic material. Furthermore, LVs are self-inactivating such that all viral transcriptional machinery is removed once genetic material is transfected into the host cell.Hemoglobinopathies require sufficiently high levels of globin genes expression for therapeutic correction, thus posing an additional challenge for successful gene therapy. Identification of critical regulatory elements required for high β-globin gene expression has resulted in feasible gene therapy options.LV vectors carrying a modified β globin gene with anti-sickling properties have been shown to be effective in both SCD and β-thalassemia murine models . Similarly, he received myeloablative conditioning with busulfan followed by infusion with the βT87Q LV-based B-globin vector. The subject did not experience any vector-related side effects nor any SCD-related hospitalizations despite discontinuing his chronic transfusion program. 30 months post transplantation he was hospitalized for management of VOC in the setting of an acute viral illness. His most recent total hemoglobin is 12.4 g/dL with no evidence of clonal dominance  toxicities attributed to the LV vector.Given ongoing safety concerns regarding oncogenesis and clonal expansion following vector insertion into the human genome, gene editing technologies remain an attractive therapeutic modality. Genome-editing therapies exploit the ability of the human genome to repair itself following double-strand breaks (DSBs). DSBs are repaired via homology-direct repair (HDR) pathways or non-homologous end joining (NHEJ). The HDR pathway is utilized to insert custom sequences into the genome via an engineered endonuclease co-delivered with an extrachromosomal repair template. These approaches employ a transient ex vivo intervention and do not result in permanent insertion of foreign DNA into the genome. Currently, Zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspersed short palindromic repeat (CRISPR/Cas) endouncleases are the systems available to make site-specific DSBs. Early proof-of-concept studies have been reported for ZFN and TALEN-mediated correction of α-thalassemia  utilize reprogramming of genes to induce the multilineage differentiation potential of mature somatic cells. This system is advantageous as it allows for screening of the ideal clone with safe integration and high gene expression profile. In vitro experiments with human iPSCs (hiPSCs) have been hampered by their inability to demonstrate terminal erythroid differentiation with mature, enucleated, β-globin- expressing erythroid cells. Utilizing optimized cell processing techniques, Rosanwo et al.  have genThis technology has been further developed via the combination of hiPSC-based cell replacement therapies with gene editing techniques for monogeneic disease correction. This approach involves generating iPSCs from patients with a disease of interest and utilizing the above mentioned gene editing strategies i.e. TALEN, CRISPR/Cas, ex-vivo to repair a mutated gene or induce expression of protein necessary to remediate disease burden  clinical trial. Results demonstrated superiority as compared to best available therapy in controlling hematocrit, reducing spleen volume and improvement in symptoms >including pruritis and night sweats. JAK2V617F allele burden decreased from baseline and declined steadily over time . Grade ≥ 3 toxicities were limited to anemia and thrombocytopenia in 2 and 3% of patients, respectively and 6% of patients developed herpes zoster infections (0% in control arm)  family. Activin expression, particularly activin A and bone morphogenic protein (BMP) 2 and 4 have been shown to have a role in the regulation of erythropoiesis  are part of a diverse group of enzymes that regulate gene expression through chromatin modification. Histone acetylation relaxes chromatin structure, thereby increasing the accessibility of transcription factors to their target genes and increases gene expression  demonstrated a significant hematologic response and resolution of grade ≥2 pruritus and splenomegaly. The combination of givinostat and HU was generally well tolerated, however premature study termination occurred in 18% of patients and grade ≥ 3 toxicity occurred in two patients  is a chromatin-modifying enzyme that maintains methylation marks on DNA throughout cell division. Methylation of DNA is important for epigenetic gene regulation and has been shown to silence genes that direct the epigenetic silencing of the HbF gene, specifically BCL11A . Lenalidomide (Len) and Pomalidomide (Pom) are newer generation analogs of thalidomide that have increased activity in multiple myeloma with an improved side effect profile. Len and Pom have been shown to reduce the transfusion burden in patients with MM and myelodysplastic syndrome (MDS) - dependent deacetylase that functions to remove acetyl groups from many histone and non-histone proteins  is the master regulator of systemic iron homeostasis. HAMP facilitates surface expression of ferroportin, an iron export protein that is largely expressed in enterocytes, hepatocytes and macrophages, and facilitates iron absorption  signaling pathway which is further modulated by total body iron availability. Hemojuvelin (HJV) functions as a co-receptor and is required to fully activate the SMAD signaling pathway . It has also been suggested to ameliorate ineffective erythropoiesis as evidenced by improved anemia, decreased alpha chain aggregates and increased proportion of mature erythroid progenitors , is a newly described erythroid regulator that is produced by erythroid precursors and has been shown to be involved in the regulation of hepcidin expression. ERFE expression is greatly increased in a murine model of β-Thalassemia which contributes to hepcidin suppression and subsequent iron overload characteristic of this disease (Kautz et al. The past 20 years have seen tremendous advancements in knowledge of the regulation of steady state erythropoiesis and in that context advancement in the understanding of the myriad molecular mechanisms leading to disordered erythropoiesis. As a consequence, multiple, novel therapeutic modalities are currently in clinical trials, many but not all, with promising results. In particular many of these new drugs target erythroid signaling, inductive a suppressive as well as the epigenetic regulation of globin gene synthesis. Furthermore, advancements in lentiviral-mediated gene therapy offer an exciting paradigm shift in treatment options as well as a promise of cure. Further clinical trials are required to expand the scope of these new therapeutic developments for clinical use.


Metabotropic glutamate receptor 5 (mGlu5) has been implicated in certain forms of synaptic plasticity and cognitive function. mGlu5 knockout (KO) mice and mGlu5 antagonists have been previously used to study the pathophysiology of schizophrenia as they have been shown respectively to display or induce endophenotypes relevant to schizophrenia. While schizophrenia presents with generalized cognitive impairments, the cognitive phenotype of mice lacking mGlu5 has so far only been explored using largely hippocampal-dependent spatial and contextual memory tasks. To address this, we used a touchscreen system to assess mGlu5 KO mice for pairwise visual discrimination, reversal learning, and extinction of an instrumental response requiring no discrimination. Furthermore, we tested the role of mGlu5 in working memory using the Trial-Unique Non-Matching to Location (TUNL) task utilizing pharmacological ablation. mGlu5 KO mice were impaired on discrimination learning, taking longer to reach criterion and requiring more correction learning trials. Performance on reversal learning was also impaired, with mGlu5 KO mice demonstrating a perseverative phenotype. The mGlu5 KO mice responded at a higher rate during extinction, consistent with this perseverative profile. In contrast, wildtype mice treated acutely with an mGlu5 antagonist (MTEP) showed no deficits in a touchscreen task assessing working memory. The present study demonstrates learning and memory deficits as well as an increased perseverative phenotype following constitutive loss of mGlu5 in this mouse model of schizophrenia. These findings will inform translational approaches using this preclinical model and the pursuit of mGlu5 as therapeutic target for schizophrenia and other brain disorders. Metabotropic glutamate receptors provide a mechanism by which the function of N-methyl-D-aspartate (NMDA) glutamate receptors can be modulated10. Aberrant NMDA receptor activity is implicated in psychiatric disorders, such as schizophrenia11. NMDA receptor antagonists induce schizophrenia-like cognitive deficits in healthy humans and rodents, including impaired cognitive flexibility12. Given their effects on glutamatergic signalling, and particularly on NMDA receptor activity, mGlu5 modulators are promising agents for treatment of psychiatric disorders, particularly the currently untreatable cognitive symptoms.The metabotropic glutamate receptor 5 (mGlu5) is an excitatory post-synaptic receptor localized largely at the periphery of the active zone, where it regulates both ionotropic receptor activity and intracellular processes via G protein-linked second messenger pathways20. However, there are problems with this approach as these findings are both occasionally inconsistent23 and the most common antagonist used, 2-methyl-6-(phenylethynyl)pyridine (MPEP), has significant off-target effects on NMDA receptors24. In contrast, mGlu5 knockout (KO) mice have clear long-term spatial and contextual learning and memory deficits as assessed by the Morris water maze (MWM) and contextual fear conditioning (CFC) paradigms25. It is thought that these impairments and other behavioural abnormalities might be a result of dysfunctional NMDA receptor signaling in these mice. These mice display a complete loss of the NMDA-receptor-mediated component of long-term potentiation in the CA1 region of the hippocampus26. In addition, mGlu5 KO mice display altered behavioural responses to the NMDA receptor antagonist MK-801 indicating that NMDA receptor function  is compromised in this mouse model27. A subset of these behavioural deficits are responsive to treatment with the clinically effective antipsychotic clozapine, supporting the predictive validity of mGlu5 KO mice in modelling schizophrenia-like behaviours27. Thus, both KO mice and antagonist-treated mice are used to model and explore the role of mGlu5 in cognition in health and disease.To explore the role of mGlu5 in cognitive domains relevant to psychiatric illness, the effect of pharmacological inhibition or genetic ablation of receptor function on rodent behavior has been commonplace. Pharmacological disruption of mGlu5 has been shown to induce spatial working and long-term memory deficits28. Cognitive flexibility, along with working memory (WM), is known to be impaired in schizophrenia30. Cognitive impairments in schizophrenia and other psychiatric disorders are not constrained to long-term or contextual memory, yet the cognitive phenotype of mGlu5-disrupted mice has so far been explored using largely hippocampal-dependent spatial and contextual memory tasks. Examination of cognitive flexibility on mGlu5-disrupted mice has so far only been examined in hippocampal-dependent contexts, where deficits have been demonstrated31. No studies have investigated working memory in mGlu5 KO mice and while a few have examined mGlu5 antagonism on working memory in rats, these suffered from potential confounding factors including off-target effects on NMDA receptors and spatial navigation impairments16.Cognitive flexibility is the ability to inhibit previously learned responses and respond to previously irrelevant information when task parameters change, typically assessed by examining perseverative behaviour on cognitive tasksTo redress this, we sought to examine the role of mGlu5 in cognitive flexibility and working memory, using clinically translatable touchscreen operant chambers. Acquisition of two-choice visual discrimination (VD) was assessed in mice with genetic ablation of mGlu5 as a measure of basic associative learning. On completion, reward contingencies were reversed, and mice were required to respond to the previously rewarded stimulus. Perseveration during completion of this reversal task was examined as a measure of cognitive flexibility. To enhance interpretation of any changes in cognitive flexibility, mGlu5 KO mice were also assessed in an extinction task. Furthermore, to assess the role of mGlu5 on working memory, C57Bl/6 mice were trained on the trial-unique delayed non-matching to location (TUNL) task. In contrast to the previously described tasks, where the outcome measures are the acquisition curves of learning and memory over many sessions, the outcome measures for TUNL are observed after successful task acquisition, when animal performance had reached a stable plateau. Working memory was assessed in C57Bl/6 mice injected with the mGlu5 antagonist 3-[ethynyl]pyridine hydrochloride (MTEP). MTEP was administered only during these probe sessions to acutely ablate mGlu5 function while working memory was being assessed without affecting baseline task acquisition. We hypothesized that in all cases, mGlu5 disruption would induce broad deficits across all task parameters.5. Genotypes were determined by PCR, from a tail biopsy. These mice were assessed on visual discrimination, reversal learning and extinction. A separate cohort of C57Bl/6 mice were obtained from Animal Resources Centre  after weaning at 4 weeks of age. These animals were assessed on VD and RL prior to the commencement of TUNL training, with other data from this cohort previously reported32.Wild-type (WT) and mGlu5 KO male mice (Grm5tm1Rod) were generated from heterozygous breeding pairs that had been maintained past generation F10 on a C57Bl/6 background3; 3–4 mice/box) from 4 weeks of age. The holding room was maintained on a 12:12 h reversed light/dark cycle at 20 ± 1 °C. At 8 weeks of age, animals were weighed daily for five days to determine a baseline weight and subsequently maintained at 85% of their free-feeding weight (FFW), with 1 g per week added until mice reached 13 weeks of age to prevent stunting. Touchscreen pre-training began at 10 weeks of age and occurred in the dark-cycle under red light. One mouse (WT) was excluded due to a failure to acquire VD. All procedures were approved by The Florey Institute of Neuroscience and Mental Health Animal Ethics Committee and were performed in accordance with the relevant guidelines and regulations of the National Health and Medical Research Council Code of Practice for the Use of Animals for Scientific Purposes.All mice were housed in standard conditions , with instructions and event recordings managed through the software Whisker Server and ABET II. Extensive apparatus methods have been published elsewhere32. Over two weeks, mice were habituated to the touchscreen chambers, trained first to initiate trials and subsequently to associate nose-poking at a stimulus on the screen with a food reward .Pre-training was conducted as described previously33. In brief, VD consisted of pairwise discrimination between a rewarded (S+) and unrewarded (S−) stimulus (contrast gratings at perpendicular orientation). The location of the S+ was pseudorandomised between trials, with no location appearing more than 3 times in a row. Designation of S+ and S− was counterbalanced within genotype groups. Touching the S+ initiated a reward sequence; whereas S− touches triggered a 5 sec timeout with the house light on, no reward provision and started a correction trial. The number of correction trials is considered a measure of perseverative behaviour as it corresponds to how long an animal takes to abandon an incorrect strategy. Correction trials consisted of representation of the previous trial until a correct response was made and were not counted towards the trial limit or number of correct/incorrect responses. Each session ended once a mouse finished 30 trials or reached a 60 min time limit, whichever came first. Criterion for the VD task was 80% correct responses for two consecutive sessions. Criteria for all tasks is listed in Supplementary Table Visual discrimination was conducted as described previouslyThe session after individual mice achieved criterion on VD they began RL. Reversal consisted of an inversion of the S+ and S− designation. All other aspects of the test were identical to VD.34. Once all mice had completed RL they commenced the acquisition phase of extinction, consisting of five consecutive sessions. A single stimulus (white square) was displayed in the centre of the screen, which when touched triggered a tone and food reward. Animals had to complete 30 trials within 12.5 mins. For the subsequent extinction phase, the protocol was identical except the food reward was omitted, leading the mice to gradually extinguish their response to the stimulus. Response rates to the stimulus were recorded for seven sessions.Extinction was conducted as described previously35. Each trial commenced with a sample phase, in which initiation triggered the display of a white square in one of five possible locations, which disappeared upon nose-poke. After a delay, a second initiation procedure was triggered by breaking the back infrared beam. This commenced the choice phase, in which the two stimuli were presented: one in the old  location and the other in the new (correct) location. Touching the correct location resulted in reward delivery, whereas a touch to the incorrect location resulted in a 5 sec timeout with the house light on and commencement of a correction trial.TUNL training: TUNL training was performed as previously describedMice were initially trained to criterion on the maximal spatial separation level, consisting of three blank locations (S3) between the correct and incorrect stimulus. Once an individual mouse reached 70% performance for two consecutive sessions, the level of its next session was reduced to S2 and then S1. Task parameters consisted of a 2 sec delay, 15 sec ITI, 5 sec correction trial ITI, 5 sec timeout and the session finishing after 45 min or completion of 36 trials.During Stage 2, mice continued to be trained on S1 but with the centre location added as a sample location. Mice were trained on S1 until group performance became stable and then moved collectively onto S0. When group performance stabilised the mice were moved onto probe trials. Task parameters were identical to those in Stage 1 with the exception of baseline training at a 0 s delay and the session finishing after 60 min or completion of 48 trials.37 This dose has been previously reported to induce impairments in Pavlovian fear conditioning31. Mice were given alternating vehicle and MTEP injection sessions for 10 days , counterbalanced across the housing groups.MTEP probe: Stable post-training TUNL performance is ideal for assessing acute pharmacology, unlike the acquisition curves observed in VD or RL tasks. MTEP  was dissolved in saline and the pH was adjusted to 7.0. Mice were given i.p. injections of 20 mg/kg MTEP or saline 10 min prior to commencement of the session during the MTEP WM probe. 20 mg/kg was chosen on the basis of previous literature suggesting this would achieve near-complete receptor occupancy for the duration of the task without inducing hyperlocomotor effects38. These are particularly appropriate for examining touchscreen data as they enable the examination of the effects of factors on a trial-by-trial basis, providing more insight than examining an aggregate ‘percentage correct’ over a session or multiple sessions. Additionally, GLLAMMs can account for non-normally distributed data, common to measures collected by touchscreens, and missing data, which occurs when mice reach criterion for a task on different days. The adjusted effect of experimental factors  on standard and correction trial counts were estimated by Poisson regression as incidence rate ratios (IRRs), or by logistic regression as odds ratios (ORs) for the TUNL experiments, and the effect sizes reported together with respective 95% confidence intervals to indicate the estimates’ precision and corresponding two-tailed p-values for the hypothesis of no effect. An IRR for the effect of genotype on correction trial count (count variable) is interpreted as a factor increase in the expected count of correction trials for a mouse of a given genotype compared to a control mouse, adjusting for all other experimental factors. For example, if the IRR was 1, we would expect equal counts of correction trials in both genotype groups, while for IRRs equal to 1.6, we would expect a 60% increase in count of correction trials in the genetically modified group compared to the control group, adjusting for all other experimental factors. Perseveration indices were calculated for each animal as total correction trial number divided by the number of incorrect responses on main trials . Total trials to criterion, total correction trials, perseveration indices and time to initiation were compared using two-tailed Mann Whitney U-tests. All statistical analyses were conducted using STATA v13IC .The associations between genotype, session, location and stimulus on correction trial count were examined at the trial’s level using a random effect regression model to estimate effect size, with individual animals treated as random effects. This was implemented using generalised linear and latent mixed models (GLLAMMs)VD involved correctly choosing the S+ paired with the reward until the association was stable. mGlu5 KO mice took significantly more trials to reach criterion , when animals are inhibiting the previously learnt association, and late (>50% correct) reversal, when animals are learning the new association, suggests that mGlu5 KO mice have deficits in both cognitive flexibility and associative learning. This is in contrast to animals with pure deficits in either associative learning, such as NR2A KO mice39, or reversal learning, such as cortical NR2B KO mice40. This perseverative phenotype was further demonstrated by the altered extinction observed in mGlu5 KO mice. Increased responding in these animals was observed throughout the extinction period, corroborating the perseverative phenotype demonstrated during early reversal learning. The extinction alteration is consistent with impairments previously reported in mGlu5 KO mice using fear conditioning6 or addictive drugs7. Notably, we did not observe impairments of working memory in WT mice subjected to mGlu5 antagonism. This suggests that while mGlu5 may be required for task acquisition, it is not needed for working memory once mice have reached a stable performance plateau. Our finding that mGlu5 is not necessary for working memory is in contrast to previous studies identifying deficits induced by MPEP16.This is the first study to report impairments in non-spatial pairwise discrimination learning in mGlu5 KO mice. These animals require both more standard and correction trials on a simple pairwise discrimination task, indicating non-specific cognitive impairment and suggesting that mGlu5 facilitates basic associative learning. This is similar to other cognitive impairment models demonstrating deficits in visual discrimination, such as NR2A KO mice31 as well as previous studies which found cognitive impairments in mGlu5 KO mice on the acquisition of memory in the Morris water maze25 and in contextual fear conditioning6. However, mice with pure deficits in associative learning, such as NR2A KO mice, manifest impairments in late – but not early – reversal learning39. Our findings of impairments in early reversal learning and alteration of extinction, as well as increased perseverative indices and correction trial number throughout all tasks, are indicative of a perseverative profile for mGlu5 KO mice. These findings are similar to those observed in mice with cortical deletion of GluN2B, and given the role of mGlu5 in potentiating NMDA receptor activity there may be a shared mechanism40. Of note is the fact that we observed increases in correction trial number and perseverative indices even during visual discrimination, before the animals had learned any associations that required inhibition. Further touchscreen testing may be useful to reveal the extent of cognitive stability vs flexibility in these animals28. These present findings indicate that mGlu5 KO mice have both impaired memory acquisition and heightened perseveration.The broad cognitive deficits of mGlu5 KO mice demonstrated in this present study raise the question of to what extent the impairments are due to difficulties in memory acquisition as opposed to heightened perseveration. On the one hand, the increase in total trials required for the animals to complete visual discrimination and late reversal learning are suggestive of memory acquisition problems. This would be supported by previous work showing that mGlu5 antagonists administered before (but not after) stimulus presentation hinder subsequent recall41, which accompanied with our lack of observation of difference in reward collection time suggests the difference is not due to altered levels of motivation. There were also no differences in latency to touch after stimulus presentation, which we would expect if the difference in initiation time was due to increased impulsivity leading to faster responses. Previous studies have not found effects of mGlu5 antagonism on impulsivity42. However, it is possible that there are impulsivity or motivational changes in mGlu5 KO mice which could be determined with more precise testing, such as with the 5-choice serial reaction time and progressive ratio tasks.Unexpectedly, we observed that mGlu5 KO mice were considerably quicker to initiate trials than WT controls. There were no differences in locomotor activity between the two groups, suggesting that this is not due to hyperlocomotion in mGlu5 KO mice. Previous studies have found no difference in motivation for a sucrose reward in mGlu5 KO mice16 may instead have been a result of MPEP impairing acquisition of a map of the environment or spatial navigation strategies, rather than affecting working memory per se. Those previous studies that used the radial-arm maze typically showed MPEP increasing reference memory errors, indicating mGlu5 antagonism induced poor acquisition of the maze. In keeping with this, MPEP has been shown to impair the stability and spatial information content of place cells43. It should also be noted that MPEP, in contrast to MTEP, has been shown to have off-target effects on NMDA receptors24. Despite MTEP having a shorter half-life than MPEP, 20 mg/kg of MTEP injected 10 min before the task commenced should have resulted in sufficient receptor occupancy throughout the session and particularly during the first half when animals complete the majority of their trials36. Our MTEP dosage in the present study was within the range used in previous behavioural studies44.Our observation that mGlu5 antagonism did not impair working memory is divergent with previous reports. One explanation could be that previous studies made use of the radial-arm maze or Y-maze, which require spatial navigation, while TUNL requires only spatial working memory. Thus, it is possible that the detrimental effects of mGlu5 antagonism reported in the previous studies46, visual discrimination48, reversal learning49 and extinction of both fear50 and appetitive49 memories. Specifically looking at working memory, MK-801 has been shown to impair performance55, but with a simultaneous decrease in performance at no delay (i.e. not in a delay-dependent manner) and frequently with hyperlocomotor side effects. Impaired performance at baseline, on other cognitive tasks, and a lack of interaction between MK-801 and delay indicates that NMDA antagonism is impairing memory formation rather than working memory per se. mGlu5 potentiation of NMDA receptors may facilitate WM in tasks requiring spatial navigation, with MPEP and MK-801 impairing performance on those tasks. In contrast, both this study and others suggest that neither mGlu5 nor NMDA receptors are necessary for WM performance in familiar environments when working memory alone is being assessed57.Given the known interaction between mGlu5 and NMDA receptors, it is worthwhile to compare our results to behavioural studies examining NMDA receptor disruption. Experiments using the NMDA receptor antagonist MK-801 show disruptions in hippocampal-dependent long-term memory39, but no impairments in extinction, while loss of NR2B from different brain regions can also impair learning, with striatal loss impairing visual discrimination and cortical loss impairing reversal learning58.More work is required to determine to what extent the cognitive impairments in mGlu5-disrupted animals are through loss of potentiation of NMDA receptors as opposed to NMDA-receptor-independent effects. In addition, it would be interesting to clarify whether NMDA-mGlu5 receptor interactions have different behavioural effects based on NMDA receptor subunit composition. For example, NR2A KO mice have both impaired discrimination and reversal learning deficits11. mGlu5, a modulator of NMDA receptors, has altered expression in patients with schizophrenia60. Schizophrenia presents with generalized learning and memory impairments, including attention, memory and cognitive flexibility12. These deficits can be assessed using the CANTAB touchscreen-based cognitive assessment battery61. Our study has demonstrated that animals with a deficit in glutamatergic signaling display deficits in memory acquisition and cognitive flexibility on touchscreen tasks analogous to those used to assess cognition in humans suffering from schizophrenia. It should be noted that developmental absence of mGlu5 may also contribute to the observed results in the present study, which may also have relevance to the pathogenesis of schizophrenia, a neurodevelopmental disorder62.The glutamatergic hypothesis of schizophrenia holds that disruption to glutamate signaling is implicated in the development of the disease. Acute administration of NMDA antagonists induce symptoms of schizophrenia in humans and animal models, and dysfunction of the NMDA receptor is hypothesized to be a key component of pathogenesis in schizophrenia32. mGlu5 KO mice are cognitively impaired but still ultimately able to learn, making them a useful psychiatric disease model in which to assess putative cognitive enhancers27.In conclusion, mGlu5 KO mice constitute a robust model of learning and memory impairments and perseverative behavior, demonstrating that mGlu5 plays a more general role in cognition than just hippocampal-dependent tasks. Touchscreen operant chambers are able to assess this in a more automatable, reproducible and translatable fashion than traditional testing and have been used to observe cognitive enhancement in wild-type animalsSupplementary Table 1


Diabetes mellitus (DM) is an endocrine disorder. In coming decades it will be one of the leading causes of death globally. The key factors in the pathogenesis of diabetes are cellular injuries and disorders of energy metabolism leading to severe diabetic complications. Recent studies have confirmed that autophagy plays a pivotal role in diabetes and its complications. It has been observed that autophagy regulates the normal function of pancreatic β cells and insulin-target tissues, such as skeletal muscle, liver, and adipose tissue. This review will summarize the regulation of autophagy in diabetes and its complications, and explore how this process would emerge as a potential therapeutic target for diabetes treatment. The term autophagy (means “self-eating”) was first depicted by Duve and Wattiaux in 1966. This isAutophagy commences by means of the formation of double-membrane vesicles, recognized as autophagosomes that swallow up cytoplasmic components. Autophagosomes combine with endosomes to form intermediate amphisomes. Amphisomes fuse with the lysosome to form autolysosomes. Autolysosomes are enriched with hydrolytic enzymes that degrade the autophagic cargo and release the degraded component into the cytoplasm of the cell for reuse Figure 1. Autophagy could be regulated by several factors such as amino acids, insulin or protein sensors . The prThe pancreatic beta cells are the major glucose regulatory cells in pancreatic islets which regulate the insulin secretions. The glucose level rises abruptly when these cells function abnormally either due to programmed cell death or by ER stress. Additionally, impairment of the autophagic pathway in pancreatic β cells also results in the development of type 2 diabetes. Recently it was revealed that “Constitutive” autophagy has a role in regulating the organization and function of pancreatic islet β-cells. For instance, β cell-specific deletion of Atg7 in type 2 diabetic mice leads to the malfunctioning basal autophagy, structural anomalies of islets and reduced insulin secretion. It has also been reported that the β cells derived from Atg7 knockout mice contains large inclusion bodies comprising ubiquitin and overexpressed p62, with reduced cell mass due to amplified apoptosis . RecentStudies conducted at the cellular level in pancreatic β cells depicts that the autophagy performs a climactic role and for this, the pathway might comprise of the LC3, PKC (protein kinase C), JNK and others. It has been depicted that autophagy performs an imperative function in shielding the β cells against hIAPP oligomerization. Treatment of β cells with human islet amyloid polypeptide (hIAPP) amplifiers autophagosome development and inhibition of autophagy enhances the cytotoxic effects of hIAPP . FurtheAutophagy in adipose tissue (AT) has also been explored in connection to type 2 diabetes as an additional key target of insulin. The formation of mature white adipocytes with unique features needs drastic cytoplasmic reorganization. Nowadays the connection between autophagy and adipogenesis has been developed. Massive autophagy is activated during adipocytes differentiation of primary mouse fibroblast. Autophagy is obligatory for lipid storage, thereafter, Atg 5 and Atg 7 is essential for the segregation of white and brown adipocytes  provides impetus to diabetes driven morbidity and mortality. Two-thirds of diabetic patients expire from heart disease or stroke globally. This has drawn attention to treating cardiovascular complications. Diabetic cardiomyopathy is one of the major complications of T2DM. It is characterized by reducing cardiomyocyte contractility, apoptosis, mitochondrial pathology, and dysfunction  that deDiabetic nephropathy (DN) has surfaced as a prime cause of end-stage renal disease and is culminating into a grave global health hazard. DN is characterized by podocyte loss due to its detachment from the glomerular membrane (GBM). Earlier evidence has demonstrated that endoplasmic reticulum (ER) stress may contribute to the development of DN  and in a mouse model of DN to study the underlying cellular mechanism of tubular autophagy. They envisaged that diabetes inhibited tubular autophagy was mediated by blocking of KCa3.1. This, in turn, was regulated by the PI3K/Akt/mTOR signaling pathway inactivation  elucidaal., 2016). Ma et al., 2016) investial., 2016 reportedThe diabetic retinopathy (DR) is one of the most predominant complications associated with diabetes  in patients associated with both types of diabetes. Diabetic neuropathy can be mediated through enhanced ROS generation, improper bioenergetics supply, apoptosis, hypercholestromia, dysfunction of mitochondria and endoplasmic reticulum, ischemia/hypoxia,nuclear factor kappa B (NF-κB)-directed neuro-inflammation, accumulation of damaged proteins and organelles in neurons and glial cells , so theSirT1 (Sirtuin 1) is an important metabolic sensor. It responds to changes in the cellular NAD+/NADH ratio. It was proposed that SirT1-mediated FOXO3a signaling reverse diabetic memory-induced epigenetic changes . TherefAutophagy is an important regulatory signaling pathway for type two diabetes mellitus. Autophagy induced by cellular stress could affect many tissues, glucose or lipid metabolism and secretion of insulin. Therefore, these conditions might lead to various chronic diseases such as diabetes complications Figure 2. T2DM itWe greatly acknowledge Dr. DS Kothari postdoctoral fellowship scheme of UGC, Government of India for funding this research.We have no conflict of interest.This review article does not contain any studies with human participants or animals performed by any of the authors.