All subjects showed relative hypometabolism in prefronto-basal ganglia-thalamic networks compared to a healthy control cohort when stimulation was switched OFF. Switching the stimulation ON resulted in differential changes in brain metabolism. Locally, volumes of activated tissue at stimulation sites (n = 6) showed a significant increase in metabolism during DBS ON compared to DBS OFF . Globally, differential changes were observed across patients encompassing prefrontal increase in metabolism in ON vs. OFF condition. Bearing in mind limitations of the small sample size, we conclude that DBS of the ventral capsule/ventral striatum for obsessive-compulsive disorder increases brain metabolism locally. Across distributed global networks, DBS appears to exert differential effects, possibly depending on localization of stimulation sites and response to the intervention.Recent approaches have suggested that deep brain stimulation (DBS) for obsessive-compulsive disorder relies on distributed networks rather than local brain modulation. However, there is insufficient data on how DBS affects brain metabolism both locally and globally. We enrolled three patients with treatment-refractory obsessive-compulsive disorder with ongoing DBS of the bilateral ventral capsule/ventral striatum. Patients underwent resting-state  Obsessive-compulsive disorder (OCD) is among the most common psychiatric disorders with a lifetime prevalence of 2–3 % . CharactFor severely affected patients that do not respond to first-line treatments such as cognitive behavioral therapy and pharmacotherapy, deep brain stimulation (DBS) for OCD has been investigated for over two decades. Despite clinical utility of the intervention and approval by the U.S. Food and Drug Administration (FDA) , the undBeyond an ongoing debate on which target may be best to treat OCD, recent approaches have suggested that clinical effects may be driven by broader network changes rather than focal brain modulation . This no15O-PET) showed that DBS ON compared to switched DBS OFF resulted in an increase of perfusion in the dorsal anterior cingulate cortex (dACC) and the basal ganglia, depending on the employed stimulation contacts of the DBS electrodes -FDG, patients waited for 30 min lying in supine position in a room with dimmed light. After that, a static image was acquired over 15 min. A low-dose CT scan for the purpose of attenuation correction was acquired prior to PET acquisition. Scans were iteratively reconstructed using a 3-D OSEM algorithm , 400 × 400 matrix, slice thickness of 3 mm). The FDG scans were then spatially normalized to MNI space  to the Fe region . The cere region ,7,9. First, we assessed FDG-metabolism in DBS OFF condition for each subject by performing a voxel-wise comparison of individual subjects with an age-matched cohort of healthy subjects that is implemented in the NEUROSTAT software . This puhttps://www.lead-dbs.org/) [http://stnava.github.io/ANTs/). Images were then normalized into ICBM 2009b NLIN asymmetric space using the SyN approach implemented in Advanced Normalization Tools. A subcortical brain shift correction was applied using LEAD-DBS if needed to attain a more precise subcortical alignment. DBS electrodes were localized within MNI space by the artifact in the postoperative image using Lead-DBS software.Volumes of interests (VOI) were selected to test how DBS affects brain metabolism locally within the electric field induced by the DBS system. To build such models of individual volumes of activated tissue (VAT), we reconstructed patients’ electrodes and electric fields in standard space using LEAD-DBS (bs.org/) . Brieflyhttp://fieldtriptoolbox.org). This created bilateral VTAs that were used as VOI in the quantitative PET analysis. We used SPSS  for non-parametric Wilcoxon signed-rank testing of dependent samples to assess differences in glucose uptake of VOIs during DBS ON and OFF conditions.Volumes of tissue activated (VTA) estimation protocol followed the one by Horn and colleagues . In brieAll three patients underwent FDG-PET imaging in both DBS ON and OFF condition. When DBS was switched OFF), all three subjects showed a relative frontal hypometabolism compared to a cohort of healthy control subjects. This hypometabolism was most pronounced in subject 1 and 2 in the medial prefrontal cortex as well as in the basal ganglia and thalamus see . There wTo assess changes in glucose metabolism induced by ongoing VC/VS-DBS, we calculated differential contrasts between DBS ON and OFF conditions. Global changes differed substantially between subjects see . Subjectp = 0.028).In a VOI analysis we specifically assessed changes in glucose metabolism in the electric fields induced by the electrodes. Interestingly, we observed an increase of glucose uptake in all subjects and VTAs see  with a sThe aim of this add-on study  was to sTo assess local changes in metabolism, we calculated individual electric fields determined by the placement of DBS leads and the individually employed stimulation settings. The resulting models form an estimation of the actual anatomical expansion of electric fields, taking into account the tissue around the electrodes and the corresponding current conduction . Notably, all subjects displayed an increase in FDG metabolism in each VAT see . Across Our finding somehow seems to contradict the former notion that DBS exerts a functional lesion in this area . HoweverLooking at the whole brain changes between ongoing and switched-off DBS resulted in a heterogeneous effect. While in one subject (subject 3), we observed only a slight increase in metabolism in the orbitofrontal and temporal cortex, the other two subjects (1 and 2) showed a partly contradicting outcome. Subject 1 revealed a wide-spread increase in glucose metabolism involving the parietal cortex as well as the medial, lateral, and orbitofrontal prefrontal cortexes with stimulation ON. Contrary to this, subject 2 showed a pronounced decrease in parietal and occipital cortical brain metabolism while FDG uptake in the thalamus was clearly enhanced with stimulation ON vs. stimulation OFF. Thus, we did not observe a clear common network effect, although increase in prefrontal activity in DBS ON condition was observed in subject 1 and partly subject 3 (but not in subject 2).The heterogeneity of the global results makes the interpretation of these findings difficult. Considering the existing literature, one possible confounder could be treatment response and thus relative decrease in OCD symptoms. Subject 1 was the only patient that clearly responded to the intervention. In fact, this subject showed an early clinical improvement that withstood a relatively low stimulation amplitude of 3.3 Volts (see ). In thiAnother possible explanation for the differential changes in glucose metabolism may result from the location of leads and the corresponding VTAs. In fact, differential changes in metabolism dependent on the employed DBS contacts have been described before . In subjongoing DBS and not with and without DBS. Hence, the DBS OFF condition does not constitute a true baseline condition.Another conceivable assumption can be derived from OFF condition measurements of glucose metabolism and clinical outcome. As presented in The major and most overt limitation of this study is the very small sample size and the consequently missing statistical group analysis. The small sample size resulted mostly from low acceptance rates of the experimental protocol within the patient sample as both exposition to radiation and the risk of relapse of symptoms during the OFF condition discouraged patients to participate to the study. Another limitation is the lack of a healthy control sample. To partly overcome this limitation, we employed publicly available healthy cohorts to compare brain activity that are used in real-life clinical FDG-PET investigations. Further limitations relate to the modelling of VTAs. Although the employed model reflects one of the state-of-the-art approaches, it is inherently an approximation of the actual anatomical expansion of the electric fields, as several influencing factors  are not taken into account. In the study design, neither patients nor investigators where blinded with regard to ON/OFF conditions. Thus, a placebo/nocebo effect cannot be ruled out. Differences in medication status may have also influenced brain metabolism, however differences between OFF and ON conditions should not be influenced by this and medication remained stable for months.Notwithstanding the small sample size, our study revealed some important insights into how DBS for OCD changes brain metabolism both locally and globally. Using state-of-art methodology to locate electrodes and model individually applied electric fields, we were able to specifically test how glucose brain metabolism is directly affected with high-frequency DBS at stimulation sites. Specifically, our results revealed heterogeneous global changes of brain metabolism including DBS-induced prefrontal increase in glucose uptake, possibly influenced by the degree of improvement of OCD symptoms as well as localization of VTAs. Locally, we observed that high-frequency DBS induced a significant increase in brain metabolism within the individually applied electric fields.


Influenza is a disease that poses a significant health burden worldwide. Vaccination is the best way to prevent influenza virus infections. However, conventional vaccines are only effective for a short period of time due to the propensity of influenza viruses to undergo antigenic drift and antigenic shift. The efficacy of these vaccines is uncertain from year-to-year due to potential mismatch between the circulating viruses and vaccine strains, and mutations arising due to egg adaptation. Subsequently, the inability to store these vaccines long-term and vaccine shortages are challenges that need to be overcome. Conventional vaccines also have variable efficacies for certain populations, including the young, old, and immunocompromised. This warrants for diverse efficacious vaccine developmental approaches, involving both active and passive immunization. As opposed to active immunization platforms (requiring the use of whole or portions of pathogens as vaccines), the rapidly developing passive immunization involves administration of either pathogen-specific or broadly acting antibodies against a kind or class of pathogens as a treatment to corresponding acute infection. Several antibodies with broadly acting capacities have been discovered that may serve as means to suppress influenza viral infection and allow the process of natural immunity to engage opsonized pathogens whilst boosting immune system by antibody-dependent mechanisms that bridge the innate and adaptive arms. By that; passive immunotherapeutics approach assumes a robust tool that could aid control of influenza viruses. In this review, we comment on some improvements in influenza management and promising vaccine development platforms with an emphasis on the protective capacity of passive immunotherapeutics especially when coupled with the use of antivirals in the management of influenza infection. Influenza viruses are highly contagious pathogens that are associated with a year-round global record reaching nearly a million morbidities and half-a-million mortalities. Four types of influenza viruses  have been identified. Influenza viruses C (isolated in pigs and humans) and D (isolated from cattle) are less common; typically, influenza virus C is associated with less severe illness ,2. On thAntigenic shift also allows influenza viruses to escape pre-existing immunity . This meThree types of vaccines against influenza are currently used worldwide including inactivated influenza vaccine (IIV), live-attenuated influenza vaccine (LAIV) and influenza virus subunit vaccine: each of which has its own advantages and drawbacks. IIV is formulated with replication-incompetent virus, due to whole pathogen inactivation usually achieved by formaldehyde treatment or split virion vaccines generated by disruption of the viral membrane . IntramuIt is worth noting that both the LAIV and IIV are cocktails of circulating seasonal influenza viruses. Mainly three viruses, i.e., A (H1N1) pdm09, A (H3N2), and the pre-determined dominant influenza B lineage (whether Yamagata or Victoria) are the constituents of the seasonal trivalent influenza vaccines (TIV). Subunit vaccines are also formulated as TIV, containing the HA of all representative vaccine strains . HoweverDue to continued burden of infection with influenza, the US CDC advocates the use of seasonal influenza vaccines in all persons >6 months prior to the winter . On the Rapid influenza virus evolution and yearly vaccine reformulations make the stockpiling of vaccines for future use a complicated issue. This subsequently delays preparation against any unforeseen epidemics. Therefore, lots of research now focuses on the development of novel broadly protective vaccine platforms, with hopes of enhancing both immunogen delivery and consequent immune response to select antigens. Some of these platforms include virus-like particle vaccines (VLP), synthetic virus vaccines, epitope vaccines, antigen-presenting cell inducible vaccines, COBRA vaccines, nanoparticle-based vaccines, and viral-vectored vaccines .Virus-like particles are non-infectious multimers of viral surface glycoproteins that have the propensity to self-assemble . VLPs arComputationally optimized broadly reactive antigen vaccines (COBRA) comprise VLPs that carry a computationally designed HA. Ted Ross’ group first generated consensus amino acid HA sequences of clade 2 highly pathogenic A (H5) involved in human infections and formulated VLPs to express this HA . H5 COBRSeveral approaches have been tried in order to generate attenuated viruses using reverse genetics technology. A suggested technique to downregulate viral protein synthesis is via biased virus codon sequences. Average codon frequency alteration can result in attenuation of virus in mice models, as shown by Fan et al., suggestive that the avian codon–biased vaccine candidate that was fitter in eggs, is good news for generation of influenza vaccines in eggs . AlternaEscherichia coli-expressed artificial recombinant protein consisting the concatenation of nine linear epitopes  of several influenza virus strains, and this vaccine has been shown to induce both cellular and humoral immunity in mice and it is envisaged as able to overcome high virus mutation rates [Epitope-based vaccines can serve both immune refocusing role as well as targeting integral virus specific epitopes. Remarkable work inspired by the stabilization of respiratory syncytial virus fusion protein (F) has shown that the relatively conserved HA stem could induce broadly neutralizing antibodies that are protective in mice and non-human primates against several virus subtypes harboring group I HAs  and group 2 HAs (H3 and H7) ,55,56. Ion rates . The epi+ DCs and plasmacytoid DCs) induced an expansion of anti-influenza virus cytotoxic T lymphocytes (CTLs) and T helper 1 (TH1) CD4+ T cells [+/Th1 T cell response [Recently, more focus is drawn to increasing the abilities of antigen-presenting cells (APCs) to efficiently involve the T-cell arm of immunity to influenza virus clearance. One of the examples is the work of Fonteneau and colleagues who showed that after exposure to influenza virus, dendritic cells (DCs)  or a parasite (Toxoplasma gondii) after immunization with nanoparticle formulation that involved a single or combination of gene-specific RNAs encapsulated in a dendrimer [Continued efforts to develop a universal influenza vaccine has driven the use of self-assembling monomeric ion-carrier molecules, called Ferritin for administration of multivalent vaccine constructs. In vivo assessment of nanoparticle-based vaccines displaying multivalent HA from 8 diverse strains of H1N1 influenza A viruses, were shown to induce broadly protective antibodies in mice, whose protection spanned strains from 1918 through 2009. The breadth of protection by the nanoparticle-induced antibodies were also shown to be more profound in comparison to the individual components of the conventional multivalent vaccine . Tao and T cells . A similendrimer . Recentlendrimer . Though endrimer .Viral-vectored vaccines platforms are designed to mimic natural infections, in that viral molecules are displayed on either similar or dissimilar virus. This approach has been shown to involve both the humoral and cellular immunity . Use of Management of ongoing influenza infection currently requires the use of antiviral drugs. Two drug classes approved for the control of influenza infections include the adamantanes (Amantadines and Rimantadines) and NA inhibitors . Whereas Adamantanes target the ion channel M2, which is involved in the release of viral ribonucleoprotein complexes in the host cell, NA inhibitors act by competitively engaging viral NA protein that is otherwise responsible for newly generated virion dissemination ,76,77,78The burden of influenza requires identification of a novel compounds that have the potential to alleviate the symptoms and reduce viral shedding. New research focuses are developing not only virus targeting antivirals but also the ones that target the host organism . For insIn 2002, Furuta et al. discovered the anti-influenza virus drug T-705, during screening of anti-influenza compounds by plaque reduction assay. T-705 was shown to have a selective index over 2000 for influenza viruses, with no detectable cytotoxicity in vitro . Trials Baloxavir marboxil is another antiviral which was first developed in Japan and was shown to act as a selective cap-dependent endonuclease inhibitor of influenza viruses’ (both A and B) polymerase subunit PA. The drug had exhibited preferable safety, tolerability, and pharmacokinetic properties in a phase I trial . The oveArbidol (Umifenovir) is another influenza-limiting drug that had previously been licensed for use in both China and Russia, for almost several decades ,117. It Ingavirin, a drug developed in Russia, adds on the current list of influenza-limiting antivirals due to its direct interference with the transportation of newly synthesized viral NP ,123,124.The need for new strategies to control influenza infections has led to the investigations of antibody therapy potential. Such an approach is based on neutralizing monoclonal antibody (mAB) expression and delivery into the host pre- or post- exposure to the pathogen . There a+ cells and phage panning to recover thirteen mABs one of which CR6261 entered clinical trials [+ cells and has completed phase IIa clinical trials [Techniques to fully recover human antibodies were further improved by Throsby et al. who used human memory Bl trials . CR6261 l trials ,131. Thil trials . DiscoveExperiments by Lu and colleagues demonstrated that the Fragment antigen-binding, F(ab’) 2 region of an equine anti-H5N1 antibody could protect mice against a lethal challenge of influenza H5N1 . Such anMost antibodies tend to be elicited against the more antigenically variable head domain of the major surface glycoprotein HA. On the other hand, the less variable stalk domain is characterized as sub-immunodominance and associated with minimal immune response ,135. ExpPassive immunotherapy has demonstrated rapid relief and life-saving capabilities in the treatment of viral infections like measles, rabies, HBV and others ,142,143.Above all, we emphasize the therapeutic capacity of novel monoclonal antibodies in combating influenza infection, and speculate that, in addition to the use of effective antivirals, passive immunotherapy against both influenza A and B viruses might be the way forward for influenza virus management amongst all classes (be it high- or low-risk) of patients. We envisage that the involvement of passive immunization will culminate in an accelerated relief through any of the mechanisms previously described, and this provides allowable time for the full activation of the adaptive immune system via the conventional antigen-presenting mechanisms. Additionally, infected persons who were passively immunized could develop a natural immunity to the specific viruses and this immunity could be long-lasting giving protection to other antigenically-matched strains . Also, tImproving vaccine delivery and efficaciousness is paramount to combat continuous circulation of epidemic influenza; however, in addition to the ongoing deployment of novel anti-viral strategies which include the highly promising immune therapies for treatment and prophylaxis are vital to limit and protect against the on-going threat of pandemic influenza.


Calcifying epithelial odontogenic tumor (CEOT) is a rare neoplasm, which accounts for < 1% of all odontogenic tumors. CEOT occurs more frequently in adults with a peak incidence in the 5th decade of life and is extremely rare in the pediatric population. We present a case of a 13-year-old girl who was found to have a mandibular CEOT. We summarize the radiological features, pathological findings, clinical management and literature review focusing on this entity in children.The online version of this article (10.1007/s12105-019-01009-1) contains supplementary material, which is available to authorized users. The calcifying epithelial odontogenic tumor (CEOT), also known as Pindborg tumor, is a rare and typically benign odontogenic neoplasm . Danish A 13-year-old female was incidentally found to have a large mandibular bone lesion during a routine dental visit. She was asymptomatic and had no complaints. There were no palpable lymph nodes on physical examination. The initial X-ray showed a lucent lesion with calcification. Orthopantomogram revealed an expansile, radiolucent lesion with scattered punctate calcifications in the mandibular body  occupying the mandibular body, extending and pushing into the cortical bone surface, associated with cortical thinning and destruction  is another option for softening the bone tissues. The difficulty is that this procedure takes longer than the current decalcification protocol, which prolongs the turnaround time by an additional 2–3 days.Although CEOT is typically benign, its behavior varies depending on the histologic features and location. Necrosis, high proliferation index assessed by Ki-67, and nuclear pleomorphism are associated with a more aggressive behavior , 32. FurOne close differential diagnosis of CEOT is adenomatoid odontogenic tumor, frequently seen in young adults with a female predilection, mostly involving the maxilla. Radiological findings can be distinguishable from CEOT when there are features of radiopaque flecks (snowflake opacities). Histological features include a thick capsule with solid tumor nests and reticular pattern with duct-like structure and polygonal cells with pale to clear cytoplasm. Spindled and columnar cells can also be seen in adenomatoid odontogenic tumor. Ameloblastoma, a common odontogenic tumor more frequently seen in young adults, also shows clinical and radiological overlap with CEOT, albeit the different morphology. The classical ameloblastoma has follicular or plexiform patterns and is composed of islands of squamous epithelial cells with palisading basal cells and reverse epithelial polarity surrounded by dense stromal tissue.AMBN (ameloblastin) gene has been found in CEOT as well as other tumors of odontogenic epithelium including ameloblastoma, adenomatoid odontogenic tumor and squamous odontogenic tumor, suggestive of a role of this mutation in tumorigenesis of the group of odontogenic tumors [PATCH1 gene mutation has also been found in both CEOT and keratocystic odontogenic tumors. However, the clinical significance of these mutations is unknown [The molecular biology of CEOT is not well understood. Mutation of c tumors . PATCH1  unknown . Demian  unknown , suggestDepending on the location, size, and expansion of the tumor, method of treatment can range from enucleation or curettage to surgical resection. Minimizing the recurrence rate largely depends on the complete resection of the tumor , 25. RecIn summary, CEOT also known as Pindborg tumor is typically a benign yet locally aggressive tumor more commonly seen in middle-aged adults, but can be seen in children, albeit rarely. The pediatric CEOT follows a benign behavior track as in the majority of the adult cases. Amyloid-like matrix is a unique component that may be associated with tumor maturation and differentiation, and possibly lower risk of malignant transformation. Meticulous handling of the specimen with careful attempt to dissect the tumor involving the soft tissue helps preserve tumor morphology for accurate diagnosis and molecular studies. Malignant tumors or metastasis are extremely rare and have not yet been reported in children. However, malignant features need to be carefully examined and excluded on a well-sampled specimen. Whether soft tissue involvement plays any role in predicting prognosis is not clear. Recurrence rate is high with incomplete resection, which warrants resection with negative margins and a long-term follow up.Below is the link to the electronic supplementary material.Supplementary material 1 (TIF 1434 KB)Supplementary material 2 (TIF 2480 KB)Supplementary material 3 (TIF 2455 KB)Supplementary material 4 (TIF 2660 KB)Supplementary material 5 (TIF 1888 KB)Supplementary material 6 (TIF 1274 KB)


Transcystic laparoscopic common bile duct exploration (LCBDE) seems safer than transductal LCBDE and is associated with fewer biliary complications. It has traditionally been limited to smaller bile duct stones however. This study aimed to assess the ability of laser-assisted bile duct exploration by laparoendoscopy (LABEL) to increase the rate of successful transcystic LCBDE in patients with bile duct stones at the time of laparoscopic cholecystectomy.Patients undergoing LCBDE between 2014 and 2018 were retrospectively analysed. Baseline demographic and medical characteristics were recorded, as well as intra-operative findings and post-procedure outcomes. Standard LCBDE via the transcystic route was initially attempted in all patients, and LABEL was only utilised if there was failure to achieve transcystic duct clearance. The transductal route was utilised for failed transcystic extraction.One hundred and seventy-nine consecutive patients underwent LCBDE; 119 (66.5%) underwent unaided transcystic extraction, 29 (16.2%) required LABEL to achieve transcystic extraction and 31 (17.3%) failed transcystic extraction (despite the use of LABEL in 7 of these cases) and hence required conversion to transductal LCBDE. As such, LABEL could be considered to increase the rate of successful transcystic extraction from 66.5% (119/179) to 82.7% (148/179). Patients requiring LABEL were however more likely to experience major complications  although none were specifically attributable to the laser intra-operatively.LABEL is an effective adjunct to LCBDE that improves the rate of successful transcystic extraction. Common bile duct (CBD) stones are reported in up to 15% of patients with symptomatic gallstones requiring laparoscopic cholecystectomy (LC) . Only a Current UK guidelines suggest that LCBDE at the time of LC as a single-staged procedure is the preferred technique for the management of gallstones with concomitant bile duct stones . AlthougTC LCBDE has traditionally been limited to smaller CBD stones that are able to pass into the duodenum after flushing or undergo extraction via the cystic duct using standard retrieval techniques. Strömberg et al. suggested that stones exceeding 5 mm in size have a much lower likelihood of TC extraction and are susceptible to impaction . As suchThe aim of this present study was to assess the ability of LABEL to increase the success rate of TC LCBDE, whilst also evaluating its safety profile in a contemporary cohort of patients.A retrospective review of a prospectively collected database of 179 consecutive patients who underwent LCBDE at a single centre between February 2014 and December 2018 was performed. All operations were performed or supervised by the senior surgeon (AI). After review of the medical records, investigation results and operative notes, all patients were assigned into two groups based on whether or not LABEL was utilised. All patients were assessed with pre-operative liver function tests (LFTs) and abdominal imaging. Patients in our institution routinely undergo pre-operative ultrasound and then intra-operative cholangiography (IOC) to identify CBD stone burden and position. In patients with very high bilirubin levels or where there is clinical suspicion of another pathology, such as a biliary malignancy, patients undergo cross-sectional imaging, usually magnetic resonance cholangiopancreatography (MRCP). In our centre, patients with acute cholecystitis and CBD stones routinely undergo LCBDE, with endoscopic retrograde cholangiopancreatography (ERCP) being reserved for high-risk surgical patients, patients diagnosed with acute cholangitis and those patients who have had cholecystitis for greater than one week, in line with international guidelines .Data collected included pre-operative demographic information, medical co-morbidity, pre-operative investigations, intra-operative findings and post-operative outcomes. Clinical presentation was classified into four groups: dilated CBD, deranged LFTs, jaundice and pancreatitis. Patients with bilirubin more than two times the upper limit of normal were classified as ‘jaundiced’ irrespective of the liver enzymes  and alkaline phosphatase (ALP)) or diameter of the CBD. Those patients with abnormal liver enzymes but bilirubin within the normal range or less than two times the upper limit of normal were classified as ‘deranged LFTs’ irrespective of CBD diameter, whereas patients with intra- or extra-hepatic duct dilatation on pre-operative imaging and normal bilirubin and liver enzymes were classified as ‘dilated CBD’. Outcomes of this study were stone clearance rates, TC exploration rate, conversion to open surgery, post-operative complications (Clavien-Dindo classification) and length of post-operative hospital stay. Length of post-operative hospital stay was chosen instead of total length of hospital stay because patients with acute cholecystitis, obstructive jaundice or pancreatitis were often admitted under the emergency surgery service and remained inpatients until their operation could be scheduled on to the next dedicated biliary operating list.TM, Optical Integrity, Florida, USA). Lithotripsy was achieved by aiming a light diode at the stone and activating the laser. Initial laser energy was set to 0.5 J (with a frequency of 20 Hz) as standard but was incrementally increased if there was failure of stone fragmentation. Fragments were either flushed into the duodenum (mainly in cases with previous sphincterotomy) or withdrawn with the Dormia basket via the cystic duct [Patients were formally consented in accordance with both local and international guidelines, including the use of LABEL, failure of stone extraction and further endoscopic or surgical procedures. At our institution, it is routine practice to attempt LCBDE with a choledochoscope via the TC route in the first instance, utilising either water flushes or the Dormia basket for retrograde retrieval. The use of LABEL was indicated when standard Dormia basket retrieval had failed via the TC route. Reasons for failure of ‘standard’ stone extraction included stone impaction within the CBD or stones too large to be retrieved via a smaller cystic duct. Cases initially failing TC extraction either underwent LABEL or were directly converted to TD LCBDE . Cases that failed TC LABEL were also converted to TD LCBDE. A laser-amenable theatre with trained staff and appropriate safety provisions in place, including a laser checklist, was used for all LABEL cases. Utilisation of the holmium laser was conducted in accordance with our previously described technique . In brietic duct . CholedoPatients requiring LABEL were compared to all those not requiring LABEL. The purpose of this comparison was to assess for laser specific complications, and as such, demographic characteristics and pre-operative investigations relating to biliary disease were also compared.p of < 0.05 was considered significant).IBM SPSS Statistics Grad Pack Version 24  was used for data analysis. Binary data was analysed with the chi-squared test for proportions and nonparametric continuous data was analysed with the Mann-Whitney U test  years, and 60 (33.5%) patients were male. Seventy-six (42.5%) patients were American Society of Anaesthesiologists (ASA) physical status classification 1, 71 (39.7%) were ASA 2, 20 (11.2%) were ASA 3, and for the remainder of patients (6.7%), no ASA classification was recorded. LABEL was utilised in 36 (20.1%) cases, and stone clearance was achieved in 177 (98.9%) patients. All procedures were completed laparoscopically with zero cases converted to open surgery. Within the whole series, 20 (11.2%) patients experienced post-operative morbidity, 17 (9.5%) of which were Clavien-Dindo I–II and 3 (1.7%) were Clavien-Dindo III–IV. There was no post-operative in-hospital mortality.TC LCBDE was planned in all 179 patients and was successfully completed in 119 of these without the need for LABEL  and respiratory disease  and proportion of higher risk patients . However, there were significantly more patients with diabetes and other endocrine disorders in the standard group . A greater proportion of patients undergoing the LABEL procedure presented with jaundice , and these patients had correspondingly higher median bilirubin levels  but had similar transaminase levels (Table p = 0.0001).When all cases utilising LABEL (n = 36) were compared to the remaining cases n = 143), baseline demographic characteristics were similar , greater size of the largest stone  and larger CBD diameter . As expected, operative times within the LABEL group were significantly longer . The LABEL group had similar rates of stone clearance (Table p = 0.248). Patients requiring LABEL also had longer post-operative stay .Patients requiring LABEL group appeared to have more complex gallstone pathology when compared with those who did not require LABEL, with more ductal calculi  although one patient in the LABEL group suffered from a mild GI bleed that was managed conservatively and another developed a small liver haematoma, also managed conservatively (Table p = 0.042). None of these complications were specifically attributable to the laser intra-operatively. Instead, they were due to two major bile leaks requiring re-intervention post-operatively . One patient had a bile leak from the duct of Luschka and re-presented after discharge with peritonitis, requiring exploratory laparotomy and washout. The other patient had a bile leak from the CBD and underwent treatment with post-operative ERCP.The overall rate of minor complications was similar between the two groups  of patients required LABEL, and these cases were more complex with a greater number and size of ductal calculi with correspondingly higher rates of failed pre-operative ERCP. However, the LABEL group had more bile leaks requiring intervention, longer operative times and longer post-operative hospital stay. These results may well be related to the greater complexity of cases within the LABEL group rather than related to laser lithotripsy itself.LCBDE, as part of a single-staged procedure, is now emerging as the preferred treatment option for patients with choledocholithiasis and gallbladder in situ, provided that the necessary expertise are available . ERCP haSeveral studies have reported the benefits of TC over TD LCBDE , 25, 26.The use of laser lithotripsy as an adjunct to LCBDE was first described in a small five-patient case series, which demonstrated efficacy even when faced with large and/or impacted ductal calculi , 17. SubTo our knowledge, this is the first Western study to highlight the utility of LABEL to increase the ability to perform TC LCBDE; our centre is now able to perform the vast majority of operations via this route. Xia et al. report a Chinese cohort with a similar increase (63.5% to 93.7%), also assuming that cases requiring laser would have required TD LCBDE had it not been available . Both rePredicting when LABEL may be required as part of LCBDE would be useful in ensuring that the correct personnel and equipment are available. Our results suggest that patients presenting with obstructive jaundice (and with higher bilirubin levels) or having failed pre-operative ERCP are significantly more likely to require LABEL. Interestingly neither transaminase levels nor CBD diameter on pre-operative imaging was associated but number and maximum size of ductal calculi were. As such, we suggest that appropriate pre-operative work up to establish this information  can assist in procedure planning, ensuring the availability of both the laser itself and of the trained auxiliary staff required for its safe and effective application.Another procedure that has recently gained traction is the so-called laparoscopic-endoscopic rendezvous procedure, which involves ERCP at the same time as LC , 32. It The key limitations of this study relate to its retrospective, non-randomised nature where patients allocated to the LABEL group were, by definition, those who had failed standard retrieval. These factors introduce significant allocation bias which decreases the comparability of the two groups, although notably both groups had similar baseline demographic and medical characteristics. Retrospectively analysing data can also lead to reporting bias. However, given the results were mostly based on binary outcomes such as success and complication rate, this is likely to be minimised in our study. Furthermore, the study was not designed, or indeed powered, to make comparisons between patients undergoing TC LCBDE with LABEL and TD LCBDE without LABEL. Instead it aimed to demonstrate that the technique was safe and effective at improving the rate of TC stone extraction. It is presumable that if a larger cohort was studied, the benefits of increased TC access would become apparent in those undergoing this technique. Further studies, with larger patient numbers and that randomise patients that initially fail TC LCBDE to either TD LCBDE or TC LCBDE with LABEL or intra-operative ERCP, are needed to conclusively prove its benefit in terms of endpoints like length of hospital stay and biliary morbidity. A final limitation is that cost-effectiveness data was not available. Future studies should consider whether laser technology is a worthwhile investment for biliary surgery; it may be most cost-effective in larger volume centres such as our own, where over one fifth of cases utilised LABEL or by sharing the equipment with other surgical subspecialties such as Urology.In terms of future directions for laser lithotripsy in biliary disease, there have been numerous studies trialling a novel laser system, the FREDDY (frequency-doubled double-pulse neodymium: YAG) laser as part of ERCP , 35. ThiOur study suggests that LABEL is an effective adjunct to LCBDE that can increase the rate of successful transcystic approach but is associated with an increased risk of major complications, though none specifically attributable to the laser itself. It can be utilised for the most complex cases, where standard retrieval has failed, with excellent results. Future research should focus on maximising the efficacy and safety of LABEL by determining the optimum laser system and operative technique.


Preventing ischemic complications while limiting bleeding risk is the cornerstone when treating patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI). Questions still persist regarding the best strategies for minimizing the risk of bleeding events when considering their actual negative impact on cardiovascular outcomes and mortality . As is oThe MATRIX  trial sought to solve this dispute ,3. The MThe co-primary endpoints selected for the MATRIX Access and the MATRIX Antithrombin were the occurrence of major adverse cardiovascular events (MACE) , and net adverse clinical events (NACE) (a composite of major bleedings [Bleeding Academic Research Consortium (BARC) type 3-5] or a MACE) up to 30-days. The primary endpoint for MATRIX Treatment Duration was a composite of urgent target-vessel revascularization, definite stent thrombosis, or NACE –4.The primary outcomes at 30-days follow-up of the three nested trials were published in 2015. The MATRIX Access showed that the use of radial access compared to femoral access was of benefit with respect to NACE , but not MACE . The reduction in NACE was driven by major bleeding unrelated to coronary artery bypass graft surgery and all-cause mortality [Recently, these findings have been reinforced by the publication of the prespecified final 1-year analysis of the whole MATRIX programme, confirming the previously reported 30-days outcomes in all respects. In the MATRIX Access, results showed a carry-over benefit for radial access compared to femoral access with respect to NACE at 1-year  mainly driven by a reduction in major bleeding events and cardiovascular death. For MACE, a trend for reduction was observed but remained formally nonsignificant  [Results from the MATRIX programme argue in clear favor of radial over femoral access for reducing adverse clinical events in patients with ACS undergoing invasive management ,4,5 and On the other hand, the comparison between the two antithrombin strategies remains difficult to interpret ,4,8. AltThus, the MATRIX programme provided prospective findings, arising from a large and multinational investigation, supporting a cause-effect relationship between bleeding mitigation strategies and mortality benefit. Considering that advanced age per se is amongst the most important bleeding risk features , every e


The accurate screening of tumor genomic landscapes for somatic mutations using high-throughput sequencing involves a crucial step in precise clinical diagnosis and targeted therapy. However, the complex inherent features of cancer tissue, especially, tumor genetic intra-heterogeneity coupled with the problem of sequencing and alignment artifacts, makes somatic variant calling a challenging task. Current variant filtering strategies, such as rule-based filtering and consensus voting of different algorithms, have previously helped to increase specificity, although comes at the cost of sensitivity.In light of this, we have developed the NeoMutate framework which incorporates 7 supervised machine learning (ML) algorithms to exploit the strengths of multiple variant callers, using a non-redundant set of biological and sequence features. We benchmarked NeoMutate by simulating more than 10,000 bona fide cancer-related mutations into three well-characterized Genome in a Bottle (GIAB) reference samples.A robust and exhaustive evaluation of NeoMutate’s performance based on 5-fold cross validation experiments, in addition to 3 independent tests, demonstrated a substantially improved variant detection accuracy compared to any of its individual composite variant callers and consensus calling of multiple tools.We show here that integrating multiple tools in an ensemble ML layer optimizes somatic variant detection rates, leading to a potentially improved variant selection framework for the diagnosis and treatment of cancer.The online version of this article (10.1186/s12920-019-0508-5) contains supplementary material, which is available to authorized users. One of the hallmarks of cancer is the accumulation of somatic genomic alterations, acquired during a cell’s life cycle and development . ThroughThe advent of NGS technologies has enabled the comprehensive detection of de novo DNA aberrations in a tumor. The continued advancements in variant detection in the last decade has resulted in a wide collection of bioinformatics tools based on different underlying statistical models. However, their results vary widely as demonstrated in several benchmarking studies –11 and nVarScan2  and VarDUnfortunately, identifying the most appropriate variant calling tool for each scenario, in addition to the adequate fine-tuning of its parameters for optimal performance, is a complex and time-consuming task –9, whichDue to the low concordance between individual tools, it is natural to address the challenge of somatic variant calling through the integration of two or more tools. Integrating the results of two or three somatic variant callers in a consensus voting filtering strategy has emerged as a standard protocol in many recent studies –24 . HowA relatively recent progression in the variant calling field to address these aforementioned problems is the application of supervised machine learning (ML). In the ML approaches, variant calling can be reduced to a classification problem, whereby each considered genomic position is labeled with its mutation status. Those base pairs that differ from the reference genome in the tumor sample, and are not present in the normal sample, will be subsequently labelled as somatic mutations.Variant calling strategies that take advantage of supervised ML can be classified into two main categories: (1) variant calling algorithms directly based on ML and (2) ensemble ML approaches that integrate multiple variant callers. SNooPer , MutatioIn the category of ensemble based variant callers it is first worth mentioning BAYSIC . While BFollowing the concept of aggregating the results from multiple variant calling algorithms in an ensemble ML layer, to yield an overall improved performance compared to the individual component tools or consensus of tools, we have developed NeoMutate. In this study, NeoMutate combines the relative sensitivities of 7 state-of-the-art variant calling tools, including MuTect2 , StrelkaFollowing the methodology used by ICGC-TCGA DREAM Somatic Mutation Calling Challenge for the synthetic dataset generation, BamSurgeon was used to spike mutations into three different well-known datasets included in the 1000 Genome Project , Support Vector Machine with linear kernel (SVMl), Gaussian Naïve Bayes (GNB), Decision Tree (DT), Random Forest (RF), Gradient Boosting Decision Tree (GBDT) and Neural Network (NN).Classifiers were comprehensively evaluated in CV experiments and on independent tests with labelled data through a robust and exhaustive set of performance measurements Table . The areNeoMutate attempts to address the inherent complexities of somatic variant calling in cancer by applying an ensemble based supervised ML approach to improve performance. Full details of NeoMutate’s methodology are described in the methods section. NeoMutate integrates different variant calling tools in an all-inclusive modular pipeline to capture a more comprehensive mutational profile and performs an ensemble ML based step for boosting detection accuracy Fig. . The worIn order to simulate synthetic ground truth somatic variants, we spiked in bona fide variants from the COSMIC database  using BANeoMutate’s performance on somatic variant detection was first evaluated through 5-fold cross-validation (CV) experiments using the reported call set from the S1 simulation experiment. The combined call set from all 7 callers consisted of 12,153 variants, 3235 (26.62%) of which were ground truth somatic mutations  and standard receiver operating characteristic (ROC) curves, for each allele frequency range, and for each variant type in order to evaluate the classifier under the different conditions Fig. . The PR In addition to VAF, tumor read depth was also evaluated as having a high impact  data from GIAB reference samples. The performance of 7 ML classifiers was comprehensively evaluated under CV and independent tests. We showed that ensemble variant calling boosted with ML, led to a substantially improved performance over standard filtering protocols, in particular decision-tree type models, which displayed a higher sensitivity and specificity balance compared to all of the individual variant callers. Additionally, NeoMutate also outperformed rule-based filtering and simple consensus approaches across a range of variant allele frequencies and mutation types. Performance of GBDT was consistent between the CV on the training data and on the independent test sets, indicating that the model was not subject to overfitting. The GBDT classifier had improved performance overall, but considerably greater performance for those variants thought to be difficult to detect (VAF < 0.05). Several studies have shown the significance of the low frequency variants in terms of their correlation with response to treatment, due to the acquisition of treatment-resistant genetic alterations in the tumor subclones , 14, 25.Each candidate variant was annotated with non-redundant biological and sequence features. The motivation behind curating the minimal amount of non-redundant features and not all possible features across all possible variant callers is based on the assumption that doing the latter would decrease the portability and scalability of the model. One of the main advantages of using an ensemble of multiple callers boosted by ML as a variant filtering strategy is that the classifiers do not rely on manually defined parameters. The ML models may automatically over time learn these parameters and attempt to capture the interplay between different features. This study incorporated 7 different state-of-the art tools, however, as new variant calling methods are developed, they can be integrated easily due to NeoMutate’s degree of modularity and adaptability. However, the benefit of adding new tools may have the greatest value if the new tool contributes to the detection of de novo true positive predictions. Since the ML framework is built upon an ensemble of embedded tools, incorporating a novel algorithm that can uniquely detect challenging variants could lead to an improved detection accuracy.The NeoMutate workflow incorporates a ML framework, where the strengths of multiple callers are exploited using a non-redundant set of biological and sequence features to boost accuracy. We have shown substantially improved performance over standard filtering protocols, specially balancing the trade-off between sensitivity to low-frequency variants and calling too many false positives, critical for an adequate decision making in personalized medicine. Moreover, ML does not rely on user-defined parameters, providing enough flexibility to deal with the common challenges present in clinical tumor samples, such as intra-heterogeneity and normal tissue admixtures. Given the unique and complex characteristics of each tumor, we demonstrate here that integrating multiple tools comprehensively in an ensemble ML layer optimizes somatic variant detection rates, leading to a potential enhancement in cancer diagnosis and treatment response.Additional file 1:Supplementary materials and methods. Detailed description of all the data generated during the study, including input data overview, in silico spiked-in variants summary, BAMSurgeon performance evaluation, used third-party tools commands. (PDF 1188 kb)Additional file 2:Comprehensive survey of ensemble ML, consensus filtering strategies and individual variant caller results across the four (S1 – S4) experiments. (XLSX 124 kb)Additional file 3:Evaluation of individual and combined variant callers on S1 experiment. Exhaustive evaluation of the composite variant callers and all possible unions and intersections between the included algorithms. (PDF 695 kb)


The ventromedial prefrontal cortex (vmPFC) is a major hub of the reward system and has been shown to activate specifically in response to pleasant / rewarding stimuli. Previous studies demonstrate enhanced pleasant cue reactivity after single applications of transcranial direct current stimulation (tDCS) to the vmPFC. Here we present a pilot case study in which we assess the cumulative impact of multiple consecutive vmPFC-tDCS sessions on the processing of visual emotional stimuli in an event-related MEG recording design. The results point to stable modulation of increased positivity biases  after excitatory vmPFC stimulation and a reversed pattern (pleasant < unpleasant) after inhibitory stimulation across five consecutive tDCS sessions. Moreover, cumulative effects of these emotional bias modulations were observable for several source-localized spatio-temporal clusters, suggesting an increase in modulatory efficiency by repeated tDCS sessions. This pilot study provides evidence for improvements in the effectiveness and utility of a novel tDCS paradigm in the context of emotional processing. The prioritized processing of emotional information within early <100 ms) [00 ms [1]To investigate the valence specificity of vmPFC and its potential to be modulated noninvasively, several studies within our lab were conducted that assessed neuronal effects of excitatory and inhibitory transcranial direct current stimulation (tDCS). In an fMRI and a separate MEG sample, participants showed valence modulation effects for passively viewed emotional scenes with increased neuronal activation for pleasant compared to unpleasant scenes after excitatory vmPFC-tDCS and vice versa after inhibitory vmPFC-tDCS . ComparaTo probe the limits and expandability of our newly developed tDCS paradigm, in this pilot study we investigated the possible cumulative effects of tDCS across 5 days of consecutive sessions with author MJ as single participant. Therefore, the event-related paradigm was similar to the MEG assessment described by Junghofer and colleagues , featuriConsistent with our prior research, we hypothesized an induction of a positivity bias after excitatory vmPFC-tDCS with increased brain activation for pleasant compared to unpleasant stimuli and vice versa after inhibitory vmPFC-tDCS by comparing neural activation after vmPFC-tDCS (Post-tDCS) with baseline activity (Pre-tDCS). Furthermore, in the case of accumulating tDCS effects, both effect patterns  were predicted to intensify across sessions. Finally, the course of Pre-tDCS data was separately investigated to test for any cumulative effects reflecting changes of vmPFC baseline activation (i.e. increasing positivity or negativity biases of baseline activity across days). Based on our previous findings on emotional scenes , we expeMJ  has given written informed consent (as outlined in PLOS consent form) to publish these case details.tDCS was applied by means of a DC Stimulator Plus (NeuroConn). The procedure was identical to previous studies ,15,27: iStimuli employed in the MEG measurement have been reported previously . They coThe study consisted of 10 daily sessions of Pre-tDCS and Post-tDCS MEG measures summing up to 20 MEG data sets. Participant MJ first passed five sessions of inhibitory stimulation and a month later, five sessions of excitatory stimulation on five consecutive weekdays each. The Post-tDCS MEG measurement always followed the tDCS in direct succession (<5 min). During MEG assessment, a passive viewing task was conducted. MJ relaxed and kept his gaze fixated on a central red dot to reduce artifacts from eye-movement. Stimuli were presented in a pseudo-random order with controlled transitions between scenes from Pleasant and Unpleasant categories which did not repeat until the complete set of 64 individual scenes had been presented. Overall, 192 stimuli (3 x 64 scenes) were presented per MEG run. Each of the 20 MEG measurements featured the same stimuli in newly pseudo-randomized orders. A single stimulus was presented for 600 ms followed by a jittered inter-trial-interval of 1,000–2,000 ms showing a gray background.k = 0.1.MEG measurements were conducted with a 275 whole-head sensor system (CTF Systems) with first-order axial gradiometers. Head movement and position were controlled via landmark coils positioned on the nasion and in both earlobes. MEG data were assessed with a sampling rate of 600 Hz. Afterwards, data were down-sampled (300 Hz) and filtered with a 0.1 Hz high-pass filter (zero-phase second-order Butterworth) and a 48 Hz low-pass filter (zero-phase fourth-order Butterworth). Trials were split into 800 ms epochs ranging from -200 to 600 ms relative to stimulus onset. Additionally, trials were baseline-adjusted by subtracting the mean of a -150 to 0 ms interval. In succession, source activation was calculated by Minimum-Norm estimates (L2-MNE) . As a soα-error inflation was established by the use of a non-parametric cluster permutation  within the excitatory stimulation condition and activation decrease per day within the inhibitory stimulation condition was analyzed. We thus applied differential weights to calculated differences [Pleasant minus Unpleasant] to test for this effect . Hypothesis 2: to investigate a cumulative stimulation effect across sessions, double differences {[Pleasant minus Unpleasant] minus [Post-tDCS minus Pre-tDCS]} were assumed to increase linearly across all five sessions within the excitatory tDCS condition, whereas inhibitory stimulation was assumed to show a linear decrease across sessions. As we were interested in cumulative Post-tDCS effects, these data were to be baseline-adjusted first for each day to control for varying Pre-tDCS activations, e.g. by additional habituation effects across days . To control for this variance, we accepted the trade-off of losing statistical power by calculating the double difference. Hypothesis 3: we finally investigated if this cumulative effect was already visible at Pre-tDCS measurements prior to daily tDCS sessions. Therefore, a correlational analysis of differences [Pleasant minus Unpleasant] at baseline (Pre-tDCS) tested for an increase of the Pleasant > Unpleasant effect across all five sessions of excitatory tDCS and vice versa (i.e. Pleasant < Unpleasant effect) across all five sessions of inhibitory tDCS . Prevention of mutation . Therebyals see ,15,27) o,27 oα-eregs.org) .r-scores, thus confirming the respective hypotheses described above. Reported p-values refer to cluster-level criterion. Importantly and further replicating findings of our previous research Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This manuscript examined a single-subject pilot study of repeated noninvasive stimulation on the ventromedial prefrontal cortex, suggesting a cumulative effect of repeated tDCS on the valence processing of emotional stimuli.The abstract summarized the primary outcomes of the study and is logical. The introduction contains three paragraphs, and the first paragraph illustrated the vital role of vmPFC on emotion processing and the different activity pattern of pleasant and unpleasant stimuli. The second paragraph demonstrated the single tDCS effect on vmPFC. It revealed an increased neuronal activation of pleasant stimuli after excitatory tDCS and vice versa after inhibitory tDCS. It then pointed out the inconsistent outcomes of consecutive tDCS stimulation and the rationale of conducting the new tDCS paradigm. The third paragraph showed the hypothesizes of the research.For the method, authors clearly described tDCS paradigm and MEG procedure. However, it seems a bit unclear about the passive viewing procedure. I am a bit confused whether the passive viewing task uses the same pictures in each tDCS sessions, or different pictures in each session? How the valence of pictures balanced? Besides, the study design without a sham tDCS group, how to prevent the placebo effect of the subject, especially under the non-blind condition?The result is answered the three hypothesizes, and the three figures also well displayed the outcomes.In the discussion part, the authors explained the relationship between three occurred cluster and tDCS stimulation, pointing out the possible changes of the neuroplasticity. Author also depicted potential limitations, especially the non-blind condition.Reviewer #2: Comments to the Corresponding AuthorThe manuscript focuses on the cumulative emotional modulation of tDCS. The theme is worth studying and the manuscript makes good reading. However, there are several issues of concern that need to be addressed:1) One big problem is the reliability and repeatability of the result. All findings were from a case, lacking of control condition that limits the interpretation and conclusions about the clinical significance of the results. The interpretation of the results is speculative, as mentioned in the limitations, these might be explained by inter-individual differences, and need to be tested in a representative sample.2) The introduction seems to be too short and too vague. The authors need to provide a more comprehensive theoretical and empirical literature review of the associations between emotional modulation and repeated tDCS to highlight the importance of repeated tDCS in emotional processing research.3) The interval between sessions of inhibitory stimulation and sessions of excitatory stimulation seems too long (one month), why?4) How long did each stimulus present? How long is the interval between stimuli? More details are needed to describe the viewing task in Procedure.5) Could you give further elaboration of results? It may be more readable and clearer to combine the analysis and result part.6) In the discussion, author mentioned “While such implicit effects might potentially explain the non-cumulative effects across sessions it is unlikely to explain the cumulation of effects”, can you give a clearer explanation?7) The authors admit the limitation of the study in that non-blinding design may cause additional influence such as attention. What ways do the authors consider can be used to examine or control this influence? Also, in the further study, single-blind or double-blind design might be more proper.In sum, I had upon carefully reading this paper, though the research topic is of interest to many researchers in emotional modulation as well as in brain stimulation, but it needs much improvement.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  26 Dec 2019Please see the attached document: Response to Reviewers.docAttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 6 Jan 2020Repeated noninvasive stimulation of the ventromedial prefrontal cortex reveals cumulative amplification of pleasant compared to unpleasant scene processing: a single subject pilot studyPONE-D-19-23160R1Dear Dr. Junghoefer,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Tifei YuanAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 13 Jan 2020PONE-D-19-23160R1 Repeated noninvasive stimulation of the ventromedial prefrontal cortex reveals cumulative amplification of pleasant compared to unpleasant scene processing: a single subject pilot study Dear Dr. Junghofer:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Tifei Yuan Academic EditorPLOS ONE


Butyrate, a product of dietary fiber and a histone deacetylase inhibitor, induces apoptosis of colorectal cancer cells; this effect of butyrate is in part mediated by its ability to hyperactivate Wnt signaling, and may in part explain the preventive action of dietary fiber against colorectal cancer. However, the mechanisms by which Wnt hyperactivation promotes apoptosis are unknown. Inactivation of the retinoblastoma tumor suppressor occurs in some cancers and can lead to context-dependent cell proliferation or cell death/apoptosis. The function of retinoblastoma protein (Rb) in normal cells is modulation of cell cycle; inactivation of Rb allows for cell cycle progression and, hence, cell proliferation. Wnt signaling is upregulated in a variety of cancers, and deregulated Wnt signaling is a key initiating event in most cases of sporadic colorectal cancer. It has been shown that Wnt signaling activated by APC inactivation can synergize with the inactivation of Rb to induce apoptosis in a manner mediated by increased TORC1 activity, leading to induced metabolic and energy stress. Rb is typically not inactivated in colorectal cancer; however, Rb is phosphorylated and deactivated during cell cycle G1/S transition. This manuscript posits that it is during this time that butyrate/histone deacetylase inhibitor-induced Wnt hyperactivation induces apoptosis in colorectal cancer cells. Thus, the inactivation of Rb in cell cycle progression may synergize with Wnt hyperactivation to induce apoptosis in response to histone deacetylase inhibitors. The hypothesis is that hyperactivation of Wnt signaling enhances colorectal cancer cell apoptosis via the interaction between upregulated Wnt signaling and inactivated Rb during cell cycle progression. This paper discusses this hypothesis and offers initial experimental approaches for testing the hypothesis. A better understanding of how histone deacetylase inhibitors induce colorectal cancer cell apoptosis through hyperactivation of Wnt signaling, and of cross-talk between repression of cell cycle and induction of apoptosis that occurs with treatment with histone deacetylase inhibitors, can assist in the development of novel therapies for colorectal cancer. Reducing morbidity and mortality from colorectal cancer requires a better understanding of cell signaling that mediates sensitivity to agents important for colorectal cancer prevention and treatment. Deregulated Wnt signaling initiates most cases of colorectal cancer and hyperactivation of this signaling by butyrate, a fermentation product of dietary fiber and a histone deacetylate inhibitor, is causally linked to apoptosis and inhibition of cell proliferation. It is unknown how Wnt upregulation causes apoptosis. Given the potential importance of fiber/butyrate for colorectal cancer prevention, and potential uses of histone deacetylase inhibitors against cancer, elucidating the mechanisms whereby Wnt hyperactivation affects cell physiology is significant.in vitroDietary fiber has been shown to be protective against colorectal cancer, and it is thought that this effect is mediated through butyrate, a histone deacetylase inhibitor that is a breakdown product of fiber Colorectal cancers can develop in individuals with higher levels of fiber intake (6-8), suggesting that butyrate resistance contributes to colonic neoplasia. To study this, a butyrate-resistant colorectal cancer cell line (HCT-R) was developed Journal of Cancer publication CBP and p300 are important in tumorigenesis, and the association between beta-catenin and CBP or p300 influences Wnt signaling Inactivation of the retinoblastoma (Rb) tumor suppressor occurs in some cancers and can lead to context-dependent cell proliferation or apoptosis Rb is typically not mutated in colorectal cancer cells The hypothesis Fig.  is that in vivo experiments utilizing appropriate mouse models.The following cell lines would be utilized to test the hypothesis: HCT-116 cells and the F5 p300 knockout line as well as the p300 rescue line in which p300 expression is restored to F5 cells .Sub-hypothesis: the temporary inactivation of Rb as part of the G1 to S transition in proliferating neoplastic colonic cells is one key point at which Wnt hyperactivation  induces apoptosis, in an energy stress-dependent manner. Further, given that this hypothesis requires cell cycle progression for Wnt activity-related apoptosis, we hypothesize competition between cell cycle arrest and apoptosis triggered by exposure to histone deacetylase inhibitors. Thus, a negative association may exist between butyrate-induced cell cycle arrest and butyrate-induced apoptosis that is dependent upon Rb inactivation during G1/S. Inhibiting the G1 to S transition It has been shown that inactivation of Rb via mutation or shRNA synergizes with upregulated Wnt activity to induce apoptosis via metabolic (energy) stress, in a manner inhibited by the mTOR inhibitor rapamycin. However, colorectal cancer cells typically express wild-type Rb, yet exhibit enhanced apoptosis associated with hyperactivation of Wnt signalingBrassica vegetables and used as a health supplement, is also in clinical trial for various forms of cancer. Cells can be treated with histone deacetylase inhibitors and apoptosis then measured as described above. mTOR activity can be repressed by treatment with rapamycin To evaluate these hypotheses, this study would determine: (a) if Rb knockdown potentiates histone deacetylase inhibitor-induced apoptosis in a Wnt activity-dependent manner, (b) whether inhibiting the G1 to S transition interferes with the ability of histone deacetylase inhibitors to promote colorectal cancer cell apoptosis, (c) whether mTOR activity is required for histone deacetylase inhibitor-induced colorectal cancer cell apoptosis, and (d) how high- and low-Wnt cell fractions influence decisions of cell cycle arrest vs. apoptosis and the role of Rb inactivation and mTOR activity in these processes. To determine whether Rb is required for histone deacetylase inhibitor-induced colorectal cancer cell apoptosis, Rb expression can be knocked out using a stably transfected Rb shRNA expression vector and cells then treated with histone deacetylase inhibitors +/- the Wnt activity inhibitor iCRT3 . Wnt reporter assays in vivo experiments be utilized to follow-up the in vitro work? The original work concerning Wnt-Rb interactions Drosophila models and it is certainly possible to adapt some of the abovementioned experimental work in that model. More pertinent for potential human utility of these studies would be experimental follow-up utilizing mouse models. The simplest approach to begin in vivo work on this hypothesis is to utilize immunodeficient mice with colorectal cancer cell xenografts, treated as above . A more powerful approach would be to utilize transgenic mouse models. The typical embryonic lethality observed with Rb-/- mice can be overcome through the use of tissue-targeted conditional Rb knockout mice APC strains of mutant mice will create in vivo models that couple Wnt hyperactivation with Rb knockout targeted to the intestine and activated at varied times during neoplastic development. These mice can be treated in an analogous fashion to the in vitro work .How could It is expected that cotreatment of colorectal cancer cells with histone deacetylase inhibitors and indole-3-carbinol in vivo work, one would expect that histone deacetylase inhibitors would enhance the apoptosis observed in Drosophila cells exhibiting Wnt hyperactivation and Rb knockdown. In xenograft mouse models, the transplanted human colorectal cancer cells would be expected to be sensitized to apoptosis induced by histone deacetylase inhibitors via Rb knockdown . This sensitization would be expected to be reversed by rapamycin or indole-3-carbinol. With respect to transgenic mouse models, intestine-targeted conditional knockdown of Rb in APC mutant mice would be expected to enhance apoptosis of the neoplastic cells, coupled to sensitization to histone deacetylase inhibitors .With respect to the proposed p300 activity is required for the competitive balance between cell cycle arrest and apoptosis in histone deacetylase inhibitor-treated colorectal cancer cells, and that butyrate resistance associated with p300 loss is in part due to aberrant Wnt activity-Rb cross-talk during cell cycle progression. Further, this manuscript hypothesizes that the accelerated G1 to S transition due to p300 deficiency interferes with mTOR activity normally observed in late G1, thus reducing energy stress and apoptosis.Colorectal cancer cells can become resistant to butyrate and other histone deacetylase inhibitors, and loss of p300 activity is associated with butyrate resistance in the HCT-116 colorectal cancer cell line Rb associates with p300 during cell cycle to modulate the G1 to S transition; it is thought that p300 inhibits Rb phosphorylation in early G1 to prevent premature entry into S phase; on the other hand, p300, and possibly CBP, stimulates Rb phosphorylation in late G1 to promote transition to S phase at the proper time G1 phase has two control points influencing cell cycle; first, the “restriction point” and a second, later “cell growth point” that is modulated by mTOR activity It is also known that Wnt signaling increases mTOR activity, which can potentiate histone deacetylase inhibitor-induced apoptosis in the presence of p300, as histone deacetylase inhibitors hyperactivate Wnt signaling (stimulating mTOR and synergizing with Rb inactivation to enhance mTOR energy stress). Interestingly, while p300 knockout cells exhibit a proliferation defect under normal culture conditions, they grow better than wild-type cells under serum starvation, exhibiting continued hyperphosphorylation of Rb (a) Determine whether the p300 status of butyrate-sensitive and butyrate-resistant colorectal cells effects levels of active vs. inactivated Rb, mTOR activity, and superoxide (a measure of oxidative stress). This study will also determine whether p300 knockout cells proliferate in serum-deprived conditions as previously reported (b) Determine whether p300 deficiency, or the balance between CBP-Wnt and p300-Wnt signaling, alters the balance between butyrate-induced cell cycle arrest vs. apoptosis to produce butyrate resistance, and whether this effect is rapamycin-sensitive, indicative of mTOR involvement, and exhibits cell type-specific effects on butyrate-induced apoptosis and proliferation arrest (c) Evaluate whether upregulation of mTOR/ TORC1 activity can reverse butyrate resistance by inducing energy stress despite premature entry into S phase.in vivo models compared to that of subsection one. However, utilization of YH249, a specific inhibitor of p300-Wnt signaling These more molecular and mechanistic experiments will be more difficult to extend to Given that (1) it is hypothesized that histone deacetylase inhibitor-induced colorectal cancer cell apoptosis is mediated by the Wnt-Rb-mTOR pathway; (2) Wnt hyperactivation (and its consequences) has CBP- and p300-mediated components (a) p300 knockout cells will exhibit reduced levels of p-RB, mTOR/TORC1 activity, and superoxide/ROS, both with respect to untreated cells and particularly after treatment with histone deacetylase inhibitors, concomitant with resistance to the apoptotic effects of butyrate and other histone deacetylase inhibitors. It is expected that under serum-deprived conditions, p300 knockout cells will exhibit a more pro-proliferative phenotype compared to wild-type HCT-116 cells under the same conditions and will thus become more sensitive to the apoptotic-inducing effects of histone deacetylase inhibitors that depend upon Rb inactivation in cell cycle progression. It is expected that in all cases, p300 rescue cells will resemble wild-type HCT-116 cells, confirming the importance of p300 in these phenomena. In addition, the observed cell type differences are expected to be reduced or abrogated by rapamycin treatment, confirming dependence on mTOR activity. Finally, energy stress will be demonstrated by a correlation between increased apoptosis and increased levels of superoxide/ROS as measured by the DHE assay.(b) Cell type-specific effects of ICG-001 and butyrate cotreatment of colorectal cancer cells on apoptosis and cell proliferation will be dependent upon the cross-talk between hyperactivated Wnt signaling and Rb inactivation during cell cycle, and will be mediated by mTOR signaling, and thus abrogated by rapamycin treatment.(c) Upregulation of mTOR/TORC1 activity will at least in part restore sensitivity to butyrate/histone deacetylase inhibitors in HCT-R and p300 knockout cells.For all subsections, it is uncertain whether colon microadenoma LT97 cells will be, to a greater or lesser extent, dependent upon the Wnt-Rb-mTOR pathway for histone deacetylase inhibitor-induced apoptosis than are colorectal cancer cells, including metastatic SW620 cells. On the one hand, LT97 cells exhibit slower proliferative dynamics than do colorectal cancer cells In all cases , normal NCM460 cells are expected to lack the Wnt-Rb crosstalk responsible for promoting histone deacetylase inhibitor-induced apoptosis.in vivo will be dependent upon Wnt hyperactivation coupled with Rb inactivation/knockdown, and will be inhibited by rapamycin, suggesting that these effects are mediated by mTOR signaling.With respect to mouse models, it is expected that the p300-Wnt inhibitor YH249 will inhibit the enhancement of apoptosis  that results from Wnt hyperactivation coupled with Rb inactivation/ knockdown. The ability of butyrate combined with an ICG-001 analog to induce cell death The main focus of this manuscript is an extension of the discovery As stated, a component of the apoptosis (and cell growth inhibition) promoted by histone deacetylase inhibitor treatment of colorectal cancer cells will be Wnt-independent. Thus, even though inhibition of Wnt signaling significantly decreases apoptosis and growth arrest induced by histone deacetylase inhibitors, apoptosis and growth arrest are not eliminated Therefore, undoubtedly, the expected cellular phenotypes will be affected by the epigenetic regulation and consequent changes in gene expression due to treatment with histone deacetylase inhibitors. However, the specific hypothesis addressed in this manuscript directly focuses on the Wnt-dependent aspects of this regulation, mediated through Rb inactivation, leading to metabolic and energy stress and apoptosis, dependent upon mTOR signaling. Wnt-independent epigenetic regulation is relevant insofar as it influences Wnt-Rb cross-talk in the manner described above.Successful completion of these proposed studies will provide a better understanding of (a) how butyrate and other histone deacetylase inhibitors induce colorectal cancer cell apoptosis through hyperactivation of Wnt signaling, and (b) cross-talk between repression of cell cycle and induction of apoptosis that occurs with treatment with histone deacetylase inhibitors. These findings can allow for (a) optimization of histone deacetylase inhibitors as preventive and therapeutic agents, respectively against colorectal cancer, and (b) provide for approaches to prevent or reverse resistance to the effects of butyrate/histone deacetylase inhibitors on neoplastic colonic cells. Countering butyrate/histone deacetylase inhibitor resistance would allow the full potential of histone deacetylase inhibitor therapy to be achieved against colorectal cancer. Thus, the findings generated by testing this hypothesis are likely to determine how synergy between Wnt hyperactivation and Rb inactivation, mediated by cell cycle-apoptosis cross-talk, can be leveraged for anti-colorectal cancer therapeutics. While not yet widely utilized for colorectal cancer treatment, histone deacetylase inhibitor colorectal cancer clinical trials have been conducted and at least one trial is ongoing. With respect to fiber and butyrate, while the effect of fiber on prevention is likely not solely due to butyrate, our work strongly suggests that butyrate is a crucial component of the preventative action of fiber. An additional objective of evaluating our hypothesis is to stimulate increased interest in the utility of histone deacetylase inhibitors in colorectal cancer prevention/therapy. In summary, a research project based on this hypothesis can impact future treatment options by demonstrating how histone deacetylase inhibitors can be more effectively utilized against colorectal cancer and may influence prevention strategies through a better understanding of the mechanisms of butyrate resistance.


Genome-wide association studies (GWAS) have been successfully implemented in cattle research and breeding. However, moving from the associations to identify the causal variants and reveal underlying mechanisms have proven complicated. In dairy cattle populations, we face a challenge due to long-range linkage disequilibrium (LD) arising from close familial relationships in the studied individuals. Long range LD makes it difficult to distinguish if one or multiple quantitative trait loci (QTL) are segregating in a genomic region showing association with a phenotype. We had two objectives in this study: 1) to distinguish between multiple QTL segregating in a genomic region, and 2) use of external information to prioritize candidate genes for a QTL along with the candidate variants.We observed fixing the lead SNP as a covariate can help to distinguish additional close association signal(s). Thereafter, using the mammalian phenotype database, we successfully found candidate genes, in concordance with previous studies, demonstrating the power of this strategy. Secondly, we used variant annotation information to search for causative variants in our candidate genes. The variant information successfully identified known causal mutations and showed the potential to pinpoint the causative mutation(s) which are located in coding regions.Our approach can distinguish multiple QTL segregating on the same chromosome in a single analysis without manual input. Moreover, utilizing information from the mammalian phenotype database and variant effect predictor as post-GWAS analysis could benefit in candidate genes and causative mutations finding in cattle. Our study not only identified additional candidate genes for milk traits, but also can serve as a routine method for GWAS in dairy cattle.The online version of this article (10.1186/s12863-019-0717-0) contains supplementary material, which is available to authorized users. Over the last decade, the development of high density single nucleotide polymorphism (SNP) arrays and next-generation sequencing (NGS) technologies have made genome-wide marker sets available in many organisms , 2. CombTo resolve these issues, we need additional information over and above association statistics. For traits with Mendelian inheritance, techniques such as homozygosity mapping and studies of recombinant haplotypes provide important clues due to the unambiguous association of at least some genotypes with phenotypic differences. For quantitative traits, no such close associations exist. However, genomic information of various types do allow relative prioritization among candidate variants. The challenges are which information to consider post-GWAS and how to combine them with GWAS statistics. Expression quantitative trait loci (eQTL) mapping can help; expression profiles as the dependent trait in a GWAS have identified causal genes in some studies . NeverthIn this study, we used an approach to separate multiple closely linked QTL in dairy cattle by fixing the lead SNP as a covariate. This approach detects QTL chromosome by chromosome, and generates a list of lead SNPs for each QTL. The method is demonstrated by application to three milk yield traits in Nordic Holstein cattle. Many previously identified loci were also confirmed here. Furthermore, we used the mammalian phenotype database to help to find the candidate genes and Variant Effect Predictor (VEP) annotations to screen for possible causal mutations.We applied a GWAS analysis approach that automatically and iteratively accounts for the effects of QTL identified in previous iteration(s), a similar approach to conditional analysis implemented in GCTA . The imp2) with the QTN in order to test whether our method introduces type I error into analysis when fixing lead SNPs detected in previous iterations as covariates  figure of previous report causative mutation of DGAT1 of the genome-wide association result milk fat yield in Nordic Holstein cattle. and Figure S2. The locuszoom figure of previous report causative mutation of DGAT1 of the genome-wide association result in milk protein yield in Nordic Holstein cattle.BOP1 was not include in USCS refFlat file [2]. (DOCX 551 kb)


OBJECTIVES/SPECIFIC AIMS: The goal for this project is to determine the feasibility of using a novel multi-photon fiber-coupled microscope to aid surgeons in localizing STN during surgeries. In order to accomplish this goal, we needed to identify the source of a strong autofluorescent signal in the STN and determine whether we could use image classification methods to automatically distinguish STN from surrounding brain regions. METHODS/STUDY POPULATION: We acquired 3 cadaveric brains from the University of Colorado Anschutz Medical Campus, Department of Pathology. Two of these brains were non-PD controls whereas 1 was diagnosed with PD. We dissected a 10 square centimeter region of midbrain surrounding STN, then prepared this tissue for slicing on a vibratome or cryostat. Samples were immuno-labeled for various cellular markers for identification, or left unlabeled in order to observe the autofluorescence for image classification. RESULTS/ANTICIPATED RESULTS: The border of STN is clearly visible based on the density of a strong autofluorescent signal. The autofluorescent signal is visible using 2-photon (850–1040 nm excitation) and conventional confocal microscopy (488–647 nm excitation). We were also able to visualize blood vessels with second harmonic generation. The autofluorescent signal is quenched by high concentrations of Sudan-black B (0.5%–5%), and is primarily localized in microtubule-associated protein-2 (MAP2)+ cells, indicating that it is likely lipofuscin accumulation in neurons. Smaller lipofuscin particles also accumulate in microglia, identified based on ionized calcium binding adopter 1 (Iba1)+ labeling. We anticipate that colocalization analysis will confirm these qualitative observations. Using 2-photon images of the endogenous autofluorescent signal in these samples, we trained a logistic regression-based image classifier using features derived from gray-level co-occurrence matrices. Preliminary testing indicates that our classifier performed well, with a mean accuracy of 0.89 (standard deviation of 0.11) and a Cohen’s Kappa value of 0.76 (standard deviation of 0.24). We are currently using coherent anti-Stokes Raman scattering and third harmonic imaging to identify different features of myelin that can be used to distinguish between these regions and expect similar results. DISCUSSION/SIGNIFICANCE OF IMPACT: Traditional methods for localizing STN during DBS surgery include the use of stereotactic coordinates and multi-electrode recording (MER) during implantation. MERs are incredibly useful in DBS surgeries, but require penetration of brain structures in order to infer location. Using multi-photon microscopy techniques to aid identification of STN during DBS surgeries offers a number of advantages over traditional methods. For example, blood vessels can be clearly identified with second harmonic generation, something that is not possible with MER. Multi-photon microscopy also allows visualization deep into tissue without actually penetrating it. This ability to look within a depth of field is useful for detection of STN borders based on autofluorescent cell density. When combined with traditional stereotactic information, our preliminary image classification methods are a fast, reliable way to provide surgeons with extra information concerning their location in the midbrain. We anticipate that future advancements and refinements to our image classifier will only increase accuracy and the potential applications and value. In summary, these preliminary data support the feasibility of multi-photon microscopy to aid in the identification of target brain regions during DBS surgeries. The techniques described here complement and enhance current stereotactic and electrophysiological methods for DBS surgeries.