Treponema have long been considered as the main causative agents because previous investigations by bacterial isolation, tissue analyses, and high molecular sequencing have persistently identified this group in BDD. However, other studies indicated that the presence of several bacteria on the lesion due to the slurry environment the cattle foot are exposed to, suggests an interdependent polybacterial nature which could also play a role in disease development and progression. Therefore, we analyzed the diversity and relationship of the diverse microbiome in BDD lesions compared to normal skin from cattle foot by using next-generation high throughput sequencing. Based on the results obtained, we concluded that the shift in microbial composition which leads to richer diversity in BDD, and the overabundance of opportunistic bacterial pathogens could be associated with BDD pathogenesis.Bovine digital dermatitis (BDD) is a foot infection known as the primary cause of lameness in cattle due to painful lesions, posing serious impacts on the productivity and welfare of affected animals. Members of the bacterial group This study analyzed the diversity and phylogenetic relationship of the microbiome of bovine digital dermatitis (BDD) lesions and normal skin from cattle foot by using 16S rRNA amplicon sequencing. Three BDD samples and a normal skin sample were pre-assessed for analysis. The Illumina Miseq platform was used for sequencing and sequences were assembled and were categorized to operational taxonomic units (OTUs) based on similarity, then the core microbiome was visualized. The phylogeny was inferred using MEGA7 . A total of 129 and 185 OTUs were uniquely observed in normal and in BDD samples, respectively. Of the 47 shared OTUs, 15 species presented increased abundance in BDD. In BDD and normal samples, Spirochetes and Proteobacteria showed the most abundant phyla, respectively, suggesting the close association of observed species in each sample group. The phylogeny revealed the evolutionary relationship of OTUs and the Euclidean distance suggested a high sequence divergence between OTUs. We concluded that a shift in the microbiome leads to richer diversity in BDD lesions, and the overabundance of opportunistic pathogens and its synergistic relationship with commensal bacteria could serve as factors in disease development. The influence of these factors should be thoroughly investigated in future studies to provide deeper insights on the pathogenesis of BDD. Bovine digital dermatitis (BDD) is known as the most important foot infection causing lameness in cattle . This seThe first reported case of BDD was in Italy in 1974 by Chelli and Mortellaro . EtiologTreponema spp. was established as the dominant pathogen in BDD lesions in Korea [Treponema spp., its polybacterial nature must also be considered in order to understand its pathogenesis [Many reports have attempted to elucidate the association of the microbiome of BDD towards disease development ,13,14,15in Korea , but an Treponema spp. by genus-specific PCR as described in the results of our previous study [This study was performed following general ethical principles and with the consent of farm owners. The collection of samples was done in Holstein–Friesian cattle in housed dairy farm, and was conducted during hoof cleaning, trimming and treatment performed by a professional veterinarian with years of expertise in the field. Inspection was done by anatomic pathological observation of grossly visible active BDD lesions on the proximal border of interdigital space characterized by the presence of ulceration, with hyperkeratosis and proliferative growth with hair-like projections as described by Zinicola et al. (2015) . For norus study . Since 1https://github.com/vsbuffalo/scythe) and Sickle (https://github.com/najoshi/sickle) programs to remove adapter sequences. The obtained 16S rRNA sequences were binned into Operational Taxonomic Units (OTUs) based on 97% identity using Quantitative Insights Into Microbial Ecology (QIIME) [The four randomly pre-assessed samples were submitted to Macrogen (Korea) for high-throughput sequencing. DNA samples extracted from lesion and normal sample biopsies were subjected to quality control by Picogreen method before library construction. By targeting the V3-V4 region of the 16S rRNA gene, libraries were constructed and were purified. Sequencing was carried out using Illumina Miseq platform . The base call binary data produced by Real-Time Analysis (RTA) were converted to FASTQ files by bcl2fastq package  and were filtered using Scythe (v0.994) ( (QIIME) . The mic (QIIME)  using a  (QIIME)  generate (QIIME) .The phylogeny of the microbiome was inferred by aligning the obtained 16S rRNA sequences of the operational taxonomic units identified using ClustalW , and theIn total, 66 samples pre-assessed from our previous research  were useThe Chao1 diversity index shows that Group B has richer species diversity compared to Group A A. The siThe maximum likelihood tree constructed using the general time-reversible model illustrated the phylogenetic relationship of all the OTUs A. The trTreponema spp. in BDD lesions and concluded that Treponema spp., was the dominant pathogen involved in BDD in Korea. Although the major abundance of Treponema spp. in lesions based on several investigations suggest that BDD is polytreponemal, the presence of other bacteria implies that they may also play certain roles in its pathogenesis.This study analyzed the diversity and phylogenetic relationship of the microbiome of BDD lesions compared to normal skin from the interdigital space of the cattle. From our previous study , we elucTreponema spp. , along with two Phorphyromonas spp. as the five most abundant bacteria in BDD. The increased abundance of these species from normal to BDD, along with others could imply that these bacteria are commensals which later progresses as opportunistic pathological agents which initiates disease development when triggered under favorable conditions.The data analyzed exhibited a change in the microbiome in BDD lesions from the normal skin sample. This altered microbiome has been previously observed by Krull et al., (2014)  and ZiniTreponema spp., as the most abundant genus. However, the dominance and diversity of species under this genus still varies between reports from different geographical locations. Moreira et al., (2018) reported that Treponema pedis is the most abundant species in BDD in Brazil, which is in accordance with Mamuad et al., (2020). However, in one study in the USA, T. denticola and T. phagedenis were the most abundant species in active and inactive lesions, respectively [T. phagedenis, which was absent in all samples in our data. This suggests that BDD microbiome has geographical and/or sample-to-sample variation.The abundance data previously presented by Mamuad et al., (2020) , to someectively . Additioectively  and Krulectively  were in Treponema are recognized as the abundant bacteria, and considered as major contributors in the development and progression of the disease, other bacterial genera have also been reported in BDD from other countries as also in this study such as Porphyromonas [Campylobacter, Acholeplasma [Peptoniphilus and Romboutsia [Carboxylicivirga mesophila and Treponema lecithinolyticum was observed as the fourth and fifth most abundant identified bacteria in BDD, respectively, which were not observed from previous studies. Further investigation is required to verify if these bacteria have actual involvement with the disease, since C. mesophila was first isolated from tidal flat sediment in Korea [T. lecithinolyticum was commonly found in human oral microbiome [Given that certain members of genus yromonas , Campyloleplasma , Peptonimboutsia . In thisin Korea , while Tcrobiome .The distance heat map between phyla and samples based on OTU count and taxonomy showed the close relationship of the observed species under phylum Spirochetes and Proteobacteria in BDD and normal skin, respectively. Phylum Bacteroidetes and Firmicutes can be associated in both groups, while Tenericutes is associated only with BDD which agrees with Nielsen et al., (2016) . In a stThe GTR tree shows the phylogenetic relationship of all OTUs present in both BDD-infected and normal skin samples, classified under 10 phyla, with Firmicutes representing the highest diversity. This large number of Firmicutes was supported by the findings of Yano et al., (2010)  and SantWe concluded that a shift in the microbiome leads to richer diversity in BDD lesions, and the overabundance of opportunistic pathogens and its possible synergistic relationship between less abundant commensal bacteria could serve as factors in disease development and progression. Spirochetes is the most abundant phylum associated with BDD in other previous studies, and in this study, we deliberated that the abundance of species on each of the observed phylum varied between reports, suggesting either geographical or sample-to-sample variation. The influence of the overabundance of opportunistic species and the synergistic interaction of the plethora of commensal bacteria should be thoroughly investigated in future studies by including additional samples categorized to varying degree of the severity of infection, not just in BDD, but also in other lameness-related foot diseases, to provide deeper insights on the pathogenesis and microbiome relationship of these debilitating diseases.


Hepatic ectopic pregnancy is an extremely rare ectopic pregnancy. This study aimed to report a case of primary hepatic pregnancy in a patient with polycystic syndrome.A 30-year-old woman presented with vaginal bleeding after 63 days of amenorrhea.The patient was initially diagnosed with liver ectopic pregnancy using abdominal ultrasound and abdominal computed tomography (CT).The patient underwent laparoscopic exploration to reconfirm the gestational sac in the liver and abdominal surgery to remove liver gestation. The postoperative review of abdominal CT and the level of serum human chorionic gonadotropin (hCG) was performed.The postoperative pathological examination revealed a fluffy tissue in the liver tissue and a blood clot. The patient's vital signs were normal, and she was advised regular follow-up after discharge from the hospital. One month later, the serum hCG level reduced to 0.32 mIU/mL (reference range 0–5 mIU/mL).If the level of beta-human chorionic gonadotropin (β-HCG) is higher than normal in women of childbearing age and no gestational sac is found in the uterine cavity, the location of pregnancy and gestational sac should be positively confirmed. Also, the possibility of ectopic pregnancy in the abdominal cavity should be considered, and the relevant imaging and biochemical examinations should be improved to avoid delay in diagnosis and treatment. If the hepatic ectopic pregnancy cannot be diagnosed and treated in time, the patient suffers a hemorrhage in the abdominal cavity. This study described the case of a patient with liver pregnancy.Ectopic pregnancy refers to the attachment of the zygote to the extrauterine cavity. It often occurs in fallopian tubes, ovaries, broad ligaments, and so on, but seldom in the abdominal cavity such as liver, spleen, or peritoneum.2A 30-year-old pregnant patient  presented at the gynecologic department with vaginal bleeding after 63 days of amenorrhea, but no abdominal/pelvic pain. The self-test urine pregnancy test was weakly positive after menopause for 35 days. The serum human chorionic gonadotropin (hCG) level showed a progressive elevation. The ultrasound (US) scan showed no gestational sac in the uterine cavity and no abnormalities in the double attachment. After menopause for 56 days, the serum hCG level was 8147 IU/L, with complaints of abdominal pain and anal bulge.2, and hemoglobin level of 131 g/L. No tenderness or rebound was observed in the upper abdomen. The US scan revealed a conspicuous amount of echogenic fluid behind the uterine cavity, a mixed echogenic mass in the hepatic area, and multiple cystic changes in bilateral ovaries  36 days after the surgery. Liver US revealed postoperative imaging changes in the liver Fig. . The pat3 based on the Medline database search. As the majority of abdominal pregnancies are implanted in the pelvis, it is rare to find ectopic implantation on the peritoneal wall of the abdomen. Although hepatic ectopic pregnancy is an exceptionally rare type of abdominal pregnancy that commonly remains undiagnosed, the possibility of an unusual location must always be a part of the practitioner's differential diagnosis. The mortality rate of hepatic ectopic pregnancy is 5 to 7 times higher than the rate observed in tubal ectopic pregnancies. Most patients with hepatic pregnancy have a history of wearing intrauterine devices, and implantation occurs on the lower surface of the right lobe of the liver. In the present case, the zygote also implanted on the right lobe, and the patient had no history of wearing intrauterine devices. Some literature raised the opinion that polycystic ovary syndrome is positively related to the occurrence of ectopic pregnancy after controlled ovarian hyperstimulation.,9 This study reported 1 case of liver ectopic pregnancy in patients with polycystic ovary without controlled ovarian hyperstimulation. Therefore, further research is needed on the risk of ectopic pregnancy in women with polycystic ovary in the absence of controlled ovarian hyperstimulation.The incidence of hepatic ectopic pregnancy is lower than that of ectopic pregnancy in other locations. Only 41 cases of primary hepatic ectopic pregnancy have been reported since 1952, According to the literature, this mechanism can be explained by the mechanism of ovarian cancer spread and metastasis.,11 Abdominal pregnancy is when the fertilized egg does not enter the uterine cavity through the peristalsis of the fallopian tube, but enters the abdominal cavity with the peristaltic and respiratory movements of the intestine. With the fluid flow in the abdominal cavity, it then grows on the dense surface of blood vessels, such as the surface of the liver and spleen.,12 Probably fatty liver and high vascularity of the hepatic parenchyma have the same effect, promoting zygote implantation on the surface of the liver and making the initial attachment in an ectopic pregnancy feasible.,13 However, due to the effect of gravity, the fertilized eggs will swim to the lowest part of the abdominal cavity. When the supine position is located, the lower surface of the right lobe of the liver is the lowest position of the abdominal cavity. It explains that the location of the ectopic pregnancy of the liver often appears on the lower surface of the right lobe of the liver.,12,14–17There is no clear evidence to explain the mechanism of liver pregnancy.,18 An abdominal US should be performed to hunt for abdominal implantation sites after a normal pelvic transvaginal US in a suspected ectopic pregnancy. US can detect fetal heartbeats, which is not only safe but also convenient for patients.,19 However, due to the thickness of the abdominal fat layer and gas in the intestine of the patient, the application of the US will also be limited. CT and magnetic resonance imaging (MRI) can reflect the tissue structure near the gestational sac, which can assist in the subsequent choice of treatment compared with US. CT with intravenous contrast enhancement can detect lesions by distinguishing density.,20 MRI can be used to diagnose ectopic pregnancy, and contrast-enhancing effects can show ring-shaped high-signal intensity in ectopic pregnancy tissues.,22 Therefore, US, CT, and MRI can provide reference and evaluation value for the diagnosis and treatment of ectopic liver pregnancy.The diagnosis of ectopic pregnancy is not simple, but the application of imaging technology can not only reduce misdiagnosis but also improve diagnosis efficiency. Although the surgical excision of ectopic pregnancies is safe, certain risks still remain. They include complications arising from the administration of a general anesthetic and injury to major abdominal structures . Due to the increased risk of maternal death, immediate surgical laparotomy and hepatectomy are commonly performed in traditional primary liver pregnancy management. The gestational sac diameter of more than 3.5 cm is a relative contraindication for methotrexate treatment. Drug treatment can be considered for the patient with suspected liver pregnancy to avoid the risk of surgery, pending further clinical data support.,27 In the present case, hepatic ectopic pregnancy was suspected before surgery, and a combination of laparoscopic surgery and open surgery approach was used to safely remove the liver gestational sac and reduce the serum hCG level to normal range.The choices of treatment for ectopic pregnancy are expectant treatment (close monitoring), drug therapy, and surgery. Therefore, the key to the successful management of an unknown-location pregnancy is high suspicion. Whether it is laparoscopic or open surgery, the postoperative serum hCG level is a common observation indicator and also one of the determinants of using methotrexate after the surgery. The data to support the conservative treatment used in the present case are still insufficient, and surgical treatment might be the first choice. Regardless of laparoscopic or open surgery, a surgical path that suits the patient's condition should be chosen. In conclusion, gynecologists should consider the possibility of hepatic implantation when faced with such a clinical presentation, although it is extremely rare.The diagnosis and treatment of these unusual abdominal pregnancies present both diagnostic and therapeutic dilemmas.The authors would like to thank our department colleagues and the devotion of this patient, and the patient has signed the informed consent form.Data curation: Yunfei Wang, Chao Zhang.Investigation: Chao Zhang.Project administration: Jing Xu.Writing – original draft: Ning Zhang, Linqing Yang, Chao Zhang, Xiaoyu Li, Jing Xu.Writing – review & editing: Xiaoyu Li, Jing Xu.Xiaoyu Li orcid: 0000-0002-0221-2336.


Background and Aim: Sepsis is a common cause of pediatric intensive care unit (ICU) admission. Sepsis-associated encephalopathy (SAE) may occur owing to brain dysfunction in those patients and may be related to impaired cerebral microcirculation. Transcranial Doppler (TCD) can be used to detect this impairment. In this study, we aimed to assess the role of TCD in prediction of SAE and mortality in patients with severe sepsis or septic shock admitted to PICU.Patients and Methods: This prospective study included 75 children admitted to PICU owing to severe sepsis or septic shock. Upon admission, all patients were subjected to careful history taking, thorough clinical examination, and standard laboratory workup. Severity of clinical illness was assessed using the Pediatric Risk of Mortality (PRISM) III score. TCD was performed on the first day of admission after the normalization of systolic blood pressure with or without vasopressors. The primary study outcome was differences in the measurement of TCD in SAE, and the secondary outcome was discharge from ICU or mortality.Results: The study comprised 45 children with SAE and 30 age- and sex-matched children without SAE. In this study, SAE patients had significantly higher pulsatility index  and resistive index  than had non-SAE patients. PI and RI showed good performance as predictors of subsequent SAE development . Non-survivors in SAE patients had significantly higher PRISM III. Receiver operating characteristic (ROC) curve analysis showed good performance of PI and RI as predictors of mortality at the end of follow-up.Conclusions: In children with SAE, cerebrovascular resistance is high and is associated with increased mortality. Sepsis contributes to 70% of pediatric intensive care unit (PICU) admission in developing countries . Brain dImpaired cerebral microcirculation and disturbed cerebral blood flow were suggested as possible mechanisms of SAE , 9. ThisBedside transcranial Doppler (TCD) may provide important diagnoses and prognoses about cerebrovascular hemodynamics in children with various disorders, for example, traumatic brain injury, intracranial hypertension, vasospasm, stroke, cerebrovascular disorders, central nervous system infections, and brain death . Three mIn this study, we aimed to assess the role of TCD in prediction of SEA and mortality in patients with severe sepsis or septic shock admitted to PICU.This prospective study was conducted at Cairo University Hospital, Cairo, Egypt. The study protocol was approved by the local ethical committee, and the legal guardians of the included children gave informed consent on admission.We included two groups: the first group comprised children in severe sepsis or septic shock with encephalopathy after confirmation of normal cerebrospinal fluid analysis, absence of primary brain pathology in head computerized tomography, or any metabolic disturbance such as electrolyte disorders, uremia, or hyperammonemia in the emergency department and prior to PICU admission. This is to eliminate the possibility of infectious meningitis, encephalitis, or encephalopathy secondary to causes other than sepsis.The other group comprised critically sick children with no encephalopathy. Sepsis was defined and classified according to the recommendations of the International Consensus Conference 2005 . SAE wasUpon admission, all patients were subjected to careful history taking, thorough clinical examination, and standard laboratory workup. Severity of clinical illness was assessed using the Pediatric Risk of Mortality (PRISM) III score .Neuroimaging studies were done to exclude cerebrovascular events. TCD was performed on the first day of admission after the normalization of systolic arterial blood pressure with or without vasopressors. We used the percentiles of systolic blood pressure to achieve a minimum of the fifth centile for age .An ultrasound machine (GE Logiq® P3) manufactured in USA with a 3-MHz TCD probe was used. The probe was directed through the temporal bone window on both sides of the skull. The temporal window was used to examine the middle cerebral artery. PI and RI were calculated as follows: PI = systolic velocity – (diastolic velocity/mean velocity); RI = systolic velocity – (diastolic velocity/systolic velocity). Every measurement on each side of the brain was repeated three times, and the highest value was considered for the analysis. The average of the two values for the two brain sides was registered. The primary study outcome was the difference in the values of PI and RI between the septic encephalopathy children and fully conscious non-septic critical children, and the secondary outcome was discharge from ICU or mortality. The images were recorded by the intensivist and reviewed by the radiologist for interpretation. The intensivist received extensive training on proper image acquisition by the pediatric radiologist. Sedation with midazolam was part of the management of ventilated patients to help reduce the intracranial pressure, and the FOUR score  was calct-test or Mann–Whitney U-test for quantitative variables and the chi-square or Fisher exact test for qualitative variables. Receiver operating characteristic (ROC) curve analysis was used to identify predictive performance of PI and RI. p < 0.05 were considered statistically significant.Data were analyzed using the Statistical Package for Social Sciences (SPSS) software, version 21 . Data were expressed as mean and standard deviations or median and interquartile range (IQR) for quantitative variables, and frequencies and percentages for qualitative variables. Comparisons between groups were performed using an independent samples p < 0.001). They also had significantly higher frequency of vasopressor use, mechanical ventilation, and deceased patients at the end of follow-up. Moreover, it was found that encephalopathic patients had significantly higher PI  and RI  than had non-encephalopathic patients. PI and RI showed good performance as predictors of subsequent SAE development  . This means that cerebrovascular resistance (CVR) increases with the deepening of coma (the decline in FOUR score). Similar results are seen in different studies that show higher CVR with more disturbance of consciousness or delirium  . SandquiIt would be better if patients were followed up after planned time period or intervention to better understand the influencing factors for changes on the cerebral hemodynamics.In cases of SAE, CVR is high and is associated with increased mortality. Future research on the possible effect of therapeutic interventions  in critically sick septic shock children for reduction of intracranial pressure and its impact on improving cerebral vasoconstriction may be beneficial. This may reduce the morbidities in septic shock survivors.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.This study was approved by the Ethics committee of Cairo University, Egypt [I-041014]. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.HA created the idea creator and supervised the work. SE edited the paper and supervised the work. AG collected and analyzed patient data and submitted the paper. RH was the radiologist who revised the sonographic data. AB was the radiologist who measured the indices. AE designed the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Indirect pathway medium-sized spiny neurons (iMSNs) in the neostriatum are well known to project to the external segment of the globus pallidus (GPe). Although direct MSNs (dMSNs) also send axon collaterals to the GPe, it remains unclear how dMSNs and iMSNs converge within the GPe. Here, we selectively labeled neighboring dMSNs and iMSNs with green and red fluorescent proteins using an adeno-associated virus vector and examined axonal projections of dMSNs and iMSNs to the GPe in mice. Both dMSNs and iMSNs formed two axonal arborizations displaying topographical projections in the dorsoventral and mediolateral planes. iMSNs displayed a wider and denser axon distribution, which included that of dMSNs. Density peaks of dMSN and iMSN axons almost overlapped, revealing convergence of dMSN axons in the center of iMSN projection fields. These overlapping projections suggest that dMSNs and iMSNs may work cooperatively via interactions within the GPe.  •Neighboring dMSNs and iMSNs were exclusively labeled with GFP and RFP•dMSNs and iMSNs formed two axonal arborizations in the GPe in a topographic manner•Axonal arborizations of dMSNs were embedded within the arborizations of iMSNs•The centers of projections from neighboring dMSNs and iMSNs were almost overlapped Biological Sciences; Neuroscience; Cellular Neuroscience The neostriatum, caudate-putamen (CPu), is the major input nucleus of the basal ganglia, which plays a key role in motor control  and receProjections from the CPu can be divided into two distinct pathways: direct pathway neurons (dMSNs) target the EP and SNr directly, whereas indirect pathway neurons (iMSNs) project to the GPe, and GPe neurons then send axons to the output nuclei via the subthalamic nucleus . In the dMSNs and iMSNs mainly project to different nuclei in the basal ganglia, but it has been reported that their outputs converge in the GPe. Although dMSNs project predominantly to the EP and SNr, several studies have revealed that dMSNs have axon collaterals in the GPe . Indeed,In the present study, we explored the precise distributions of the varicose fibers of dMSNs and iMSNs in the mouse GPe. We injected an adeno-associated virus (AAV) vector into the CPu of dopamine receptor D1 (Drd1)-Cre transgenic mice  and labeIn order to label neostriatal neurons of the direct and indirect pathways with different fluorescent proteins, we applied the Cre-dependent recombination system with an AAV vector developed in this study and Cre-expressing transgenic mice. We first validated the specificity of Cre recombinase expression in dMSNs of Drd1-Cre transgenic mice using double immunofluorescence staining for Cre and chemical markers for dMSNs and iMSNs. MSNs are inhibitory GABAergic neurons and account for over 95% of neostriatal neurons . dMSNs aWe developed an AAV vector that expresses palmitoylation site-attached GFP (pG) or palmitoylation site-attached RFP (pR) in the presence or absence of Cre recombinase, respectively C. Focal To visualize axon fibers with high sensitivity, we performed immunoperoxidase staining for GFP and RFP in mouse brain sections (n = 3 mice). In the CPu, strong immunoreactivities for GFP and RFP were observed in cell bodies, dendrites, and axons A and 2G.Consistent with a previous report , we obseWe investigated the distributions of GFP+ and RFP+ axon fibers in the GPe by AAV vector injections into the CPu of Drd1-Cre mice. We first quantified the number of GFP+ and RFP+ MSNs in each mouse and superimposed the locations of cell bodies in the parasagittal plane . The numAfter tracing GFP+ and RFP+ axon fibers in separate sections, two adjacent sections were superimposed in the parasagittal plane  and S2. We subsequently analyzed the axonal distributions of dMSNs and iMSNs in the GPe. Both RFP+ and GFP+ fibers formed two axonal arborizations in the parasagittal plane, although the two arborizations were not clear in the trace image of mouse #3 . To deteIn addition to the target centers of MSNs in the GPe, we analyzed the projection area of GFP+ and/or RFP+ axon fibers in the GPe, focusing on the area where the axons were distributed rather than on axon density. We divided the GPe into 40 μm × 40 μm boxes and classified individual boxes based on the presence of GFP+ and/or RFP+ varicose fibers A. In theThe fact that neighboring dMSNs and iMSNs sent axons to the nearby subfield in the GPe suggests topographical organization of striatopallidal pathway. We then investigated the relationship between the positions of the infected MSNs in the CPu and the distributions of axon fibers in the GPe. The centers of the infected soma distributions were calculated from Moreover, the striatopallidal circuit topography is known to be based on calbindin (CB) immunoreactivity, where MSNs in the CB-rich region in the CPu project to the CB-rich region in the GPe, and vice versa ; the GPeIn this study, we characterized the axonal projection patterns of dMSNs as well as iMSNs in the GPe by using the AAV vector that selectively labels neighboring dMSNs and iMSNs with different fluorescent proteins. Both dMSNs and iMSNs formed two axonal arborizations in the rostral and caudal parts of the GPe and showed topographical projections in the dorsoventral and mediolateral planes. The axon fibers of dMSNs displayed a spatially limited distribution within the iMSN-targeted subfield in the GPe . This ovAlthough we analyzed the projections of dMSNs and iMSNs to the GPe using anterograde labeling with the AAV vector, we did not confirm the projections by retrograde tracing techniques in the present study. As iMSNs project to a wider range of the GPe and only a portion of dMSNs send axon collaterals to the GPe , more iMIn Drd1-Cre transgenic mice, Cre recombinase is specifically expressed in dMSNs, enabling labeling of dMSNs with GFP by the flip-excision (FLEX)  switch-cTopographical projections of dMSNs and iMSNs to the GPe were clearly observed in the dorsoventral and mediolateral planes, a finding that was in good accordance with that of a previous study demonstrating similar topographical projections of a mixture of dMSNs and iMSNs . Using tThe GPe is known to be divided into two regions—CB-rich and CB-poor regions . We haveThe present study demonstrated that dMSNs and iMSNs formed two axonal arborizations in the same regions of the GPe. These overlapping projections may form the structural basis for cooperative function of dMSNs and iMSNs, consistent with the emerging model of the basal ganglia . It has Our present findings of convergence of dMSN axons in the central area of iMSN projections in the GPe extend the classical model of the basal ganglia. In the classical model, dMSNs enhance the activity of thalamic neurons through output nuclei (EP and SNr) and facilitate desired actions, whereas iMSNs suppress competing movements by inhibiting the thalamus . The axoIn this study, the distribution of axon fibers in the GPe was analyzed using bright-field staining for GFP or RFP in 20-μm-thick adjacent sections, but all fibers were not visualized and quantified. In order to accurately estimate the length and distributions of GFP+ and RFP+ fibers in the GPe, the fibers should be fully reconstructed in 3D. Owing to the high density of the projecting fibers to the GPe, it is laborious to perform a full 3D reconstruction with thin sections, and it would be more efficient to observe the fibers three-dimensionally with thicker specimens, such as slices and hemispheres, using a tissue-clearing method.Furthermore, we analyzed the projections of dMSNs and iMSNs using only Drd1-Cre mice but not Drd2- or A2a-Cre mice, which express Cre recombinase specifically in iMSNs in the CPu. The use of these strains would have led to more convincing conclusions about the overlapping projections of dMSNs and iMSNs to the GPe.Finally, and most importantly, the current technique does not allow us to determine whether dMSNs and iMSNs target the same or different neurons in the GPe. Therefore, in order to elucidate the functional significance of the overlapping projections revealed in this study, it is important to identify the types of GPe neurons that receive synaptic inputs from dMSNs and/or iMSNs in future studies.h-hioki@juntendo.ac.jp).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hiroyuki Hioki (All unique biological materials generated in this study are available from the lead contact with a completed materials transfer agreement.All data and custom scripts used in this study are available upon request.All methods can be found in the accompanying


Conventional cancer chemotherapy presents notable drug side effects due to non-selective action of the chemotherapeutics to normal cells. Nanoparticles decorated with receptor-specific ligands on the surface have shown an important role in improving site-selective binding, retention, and drug delivery to the cancer cells. This review summarizes the recent reported achievements using nanostructured coordination polymers (NCPs) with active targeting properties for cancer treatment in vitro and in vivo. Despite the controversy surrounding the effectivity of active targeting nanoparticles, several studies suggest that active targeting nanoparticles notably increase the selectivity and the cytotoxic effect in tumoral cells over the conventional anticancer drugs and non-targeted nanoparticle platform, which enhances drug efficacy and safety. In most cases, the nanocarriers have been endowed with remarkable capabilities such as stimuli-responsive properties, targeting abilities, or the possibility to be monitored by imaging techniques. Unfortunately, the lack of preclinical studies impedes the evaluation of these unique and promising findings for the translation of NCPs into clinical trials. Nanostructured Coordination Polymers (NCPs) represent a versatile class of nanomaterials based on the coordination of structural and functional organic ligands with metal ion nodes. The rich chemistry and infinite combination of different binomial metal-ligand units make these hybrid materials a fascinating class of nanomaterials with interesting applications. The self-assembly of NCPs with well-defined compositions and homogeneous sizes is a challenging area of growing interest. The chemical flexibility of these materials allows for systematically controlling and tuning their physicochemical properties and endowing them with interesting optical, magnetic, electronic, and catalytic properties ,2. The nTraditionally, the term NCPs has been used for amorphous nanoparticles and the crystalline and porous counterpart have been named as nanostructured metal-organic frameworks (NMOFs) . Even thThe special features and capabilities of NCPs have positioned them to be used in medical applications. These nanomaterials have special ability to integrate by physical encapsulation or chemical entrapment of a wide range of active species . As a geThe use of nanoparticles in cancer treatment has reduced the systemic toxicities and improved the pharmacokinetics of anticancer drugs through passive or selective targeting to tumors with the subsequent local delivery of the therapeutic agents. The fine tune of nanoparticles sizes, surface passivation, and surface coating with specific targeting molecules for the detection of tumoral cells have increased the anticancer action of well know anticancer drugs and the design of new nanodrugs. The ability of most of the nanoparticles to passively target tumors taking advantage of the leaky vasculatures in the tumoral area through the enhanced permeability and retention (EPR) effect, or the interaction of targeting molecules with specific receptors in the membrane of tumor cells (active targeting) has allowed increasing the therapeutic effect of the drugs by minimizing systemic toxicity or other side effects. Moreover, the ability of some nanoparticles to include different drugs in the same nano-construct with the possibility to modulate the release profiles can induce or maximize drugs′ synergistic effect.An extensive number of publications and review articles have shown the progress in improving drug delivery pharmacokinetics and altering the drugs′ biodistribution by preferentially accumulating the nanoparticles in diseased tissues. The principal efforts have been focused on finding new strategies for incorporating different drugs into the coordination polymer matrix by controlling the drug release profiles and providing multifunctionality to the nano-formulation. The potential multifunctionality of these nano-systems and their chemical flexibility open new possibilities for their use as smart drug delivery systems, bioimaging probes, or a combination of both (theranostic applications). The increasing number of in vitro and in vivo studies concerning the use of NCPs for therapy and diagnosis in cancer treatment helps to understand their mechanism of action and their interaction with biological environments. As a result of these studies, a series of limitations and challenges have been highlighted as critical points for the clinical translation. Like many nanoparticles used in nanomedicine for cancer treatment, NCPs have the advantage of targeting cancer by simply being accumulated and entrapped in tumors by the EPR effect (passive targeting), which was caused by leaky angiogenetic vessels and poor lymphatic drainage . Some stThere are several examples of successful cancer diagnostic and therapeutic nanoparticles, and many of them have rapidly moved to clinical trials . TherefoFrom 2005, when the first publications concerning the use of NCPs for biomedical applications were described, numerous articles were published showing the incorporation of multiple functionalities through the rational selection of specific metal ions and organic linkers. Various NCPs demonstrated their ability to co-encapsulate and deliver drugs and/or imaging agents in a controlled manner, which is difficult to achieve with other nano-formulations. Even though NCPs are not hollow or open-framework structures, their tunable matrix composition facilitates encapsulation of different functional species. The encapsulation usually proceeds through two distinct strategies: (1) using the drug as a building block integrated into the NCPs′ polymeric framework ,30,31,32Coordination bonding has emerged as a versatile method to prepare nano-complexes by assembling drugs into polymeric nanoparticles. Most of the studies showed that different metals can form coordination bonds with drugs such as daunorubicin or doxorubicin (DOX) , methotrMany of the current nanomedicines, used for solid tumor treatment, accumulate in tumors due to the EPR effect. A thorough knowledge of the physical properties  and chemical attributes of nanoparticles facilitates their application in biomedicine . SpecifiIn this review, we will summarize the most recent strategies for coating and surface functionalization of NCPs designed for passive and active targeting in cancer treatment.Since resistance to the platinum chemotherapeutic agents is a major limitation for the successful treatment of many cancers, development of novel strategies to overcome intrinsic and acquired resistance to chemotherapy is of critical importance for the effective treatment of different types of cancers. In the case of NCPs, the exploration of non-classical mononuclear or polynuclear platinum(II) compounds, platinum(IV) prodrugs, or photoactivatable platinum (IV) complexes has afforded an intense number of research works including supramolecular self-assembled nanostructures, metal-organic frameworks, and diverse coordination polymer-based nano-formulations for developing the next generation of platinum cancer drugs.νβ3 integrin, which provided actively targeted cancer in vitro in the overexpressed HT-29 cell line. Although Pt (IV) prodrug remains inactive in physiological conditions, once inside the cells, the Pt (IV) complexes can become the pharmacologically active Pt (II) species by the action of reducing agents  present in the intracellular environment [Since 2008, Lin and coworkers have been developing a series of platinum-based NCPs nano-constructs to deliver platinum-based chemotherapeutics. Although the first publications were related to the synthesis, physicochemical characterization and study of NCPs’ cytotoxic effects ,59,60. Iironment . The resApart from the silica coating, the stabilization with lipid coatings is one of the most common methodologies for conferring physical and chemical stability to NCPs. The lipid coating allows functionalizing the surface by targeting molecules such as anisamide (AA) to enhance cellular uptake and cytotoxicity in human cell lung cancer cells , or usinOther nano-constructs have been designed for the co-delivering of chemotherapeutics and siRNAs to eradicate tumors of cisplatin-resistant cancer by overcoming intrinsic and acquired resistance to chemotherapy ,65. The Some examples of NCPs designed to target specific organs were published recently. In these systems, the use of precise biomolecules for improving the nanoparticles accumulation in the site of action has allowed the efficient delivery of therapeutics. Zhao and coworkers designed a cisplatin prodrug bearing Zn-based NCPs, which were coated with polyethylene glycol (PEG) and decorated with alendronate (ALN), which is a bone-seeking moiety . The resCancer immunotherapy is an interesting field in which the nanotechnology can afford different solutions . It has Apart from the ability of NCPs to encapsulate or integrate metal-based anticancer drugs  as building blocks, these nanoparticles have demonstrated their capacity for encapsulating small chemotherapeutics by physical entrapment into the NCPs′ matrix or using the coordination ability of some organic drugs to be included as bridging ligands in the coordination polymer network. As previously mentioned, the mode of drug incorporation will determine the release profile of the nano-construct . UsuallyThe lability of coordination bonds in NCPs facilitates the fabrication of nano-systems with responsive properties that may be used to improve the stability in physiological environments and control of targeted delivery. This pH-response behavior is of special relevance due to the pH changes in cancer cells and tumoral environments. Taking advantage of these properties, a vast number of research works have been developed to assure a rapid and quantitative release in tumors subject to pH regulation ,37.Despite the therapeutic importance and targeting properties of drug-contained NCPs in vitro ,79,80, fAnother example of NCPs effective in the combination of chemotherapy and PDT was reported by Liu and coworkers recently. The authors presented a type of NCPs formed by the coordination of Cu (II) ions with the banoxantrone (AQ4N) and doxorubicin . The inhAlthough PDT is proposed in combination with chemotherapy, various NCPs designs have been developed to deliver PDT agents to tumors. However, only a few of them achieved a clinically relevant anticancer efficacy. Apart from the photosensitizer-containing porous metal–organic framework (MOF) nano-systems ,85, some1O2), which is a specie able to provoke cell apoptosis/necrosis and induce antitumor immunity [PDT is an effective anticancer procedure that involves the administration of a tumor-localizing photosensitizer (PS) that can be activated by light activation to generate highly cytotoxic reactive oxygen species (ROS) in situ. Usually the ROS production is related to generating singlet oxygen -phthalocyaninatozinc(II) (TPZnPc) photosensitizer coordinated with zinc ions. The preformed nanoparticles were coated with a lipid bilayer containing 1,2-dicarboxylic-cyclohexane anhydride modified lysyl-cholesterol (DLC) that promotes tumor cellular uptake through an electrostatic interaction induced by the mildly acidic tumor microenvironment . These Ntive PDT .2O2 to in situ generated oxygen in order to further enhance PDT by overcoming the hypoxia-associated resistance. This design showed an interesting concept of multifunctional NCPs for combining drug delivery properties with a PDT effect, down-regulation of antiapoptotic proteins, and the modulation of the unfavorable tumor microenvironment simultaneously.A more complex and multifunctional nano-construct has been developed by Chen and coworkers using DNA-based NCPs obtained by the coordination of Ca (II) ions and AS1411 DNA G quadruplexes functionalized with chlorine e6 (Ce6) photosensitizer, hemin, and iron-containing porphyrin . FurtherThe use of NCPs has opened new avenues in the design of new therapeutic tools and strategies to treat many cancers. Different new approximations try to solve problems related to the physiology of tumor extracellular matrix (ECM) to improve the tumor retention and penetration of therapeutic agents , the use2Ir(Hdcbpy) and Dy(OOCCH3)3 hybridized Au nanocages (AuNC@CPs) that offer excellent photothermal, photoacoustic, and magnetic properties in solution. These special features make AuNC@CPs interesting materials for near-infrared (NIR)-driven photothermal therapy (PTT) guided by photoacoustic (PA) and magnetic resonance (MR) imaging in vivo [The development of hybrid nano-systems combining coordination polymers with other materials has increased the pool of materials with biomedical applications using novel approximations with high potentiality. Thus, we can found some examples like the coordination polymer derived from (pq) in vivo . Other i in vivo .Some of the recent publications have shown interesting multifunctional nanoplatforms, such as that reported by Liu and coworkers, which detailed a biomimetic nanoprobe based on a pH-responsive porous NCPs for activated fluorescence imaging and targeted drug delivery . The repAnother interesting example of multifunctional and synergetic tumor-targeted nanoparticles were reported by Pan and coworkers. The authors reported a chemo-photothermal combined therapeutic nanoplatform based on PEGylated borate-coordination-polymer coated with polydopamine (PDA@CP-PEG) . The nanAs observed previously, there are few examples of active targeting for cancer treatment in the case of NCPs. However, currently, there are many types of nanoparticles at an early design step that may progress in the future to preclinical development for cancer imaging and therapy. A particular version of these nanoparticles is the targeting-active nanoparticles. In this scenario, there is still plenty of room for improvement.The local delivery of a specific drug, or a combination of synergic drugs, can lead to cell killing in a very specific tumor in a short period of time. The development of more effective and selective therapies would avoid surgery and/or chronic chemotherapy treatments by increasing the quality and life expectancy of the patient. Thus, more efforts should be put in developing new types of NCPs’ coatings  to increase the physicochemical stability of the nanoparticles in physiological environments, and design new manners to decorate the nanoparticles’ surface with specific targeting molecules  to adequately treat specific cancer types. There are important factors that should be revised and studied such as the performance of PEG-coated nanoparticles (PEG size and its grafting density) in order to design and apply for optimum circulation times and tumor cell uptake. In the same way, it is important to design new targeting ligands to avoid macrophage recognition and faster clearance compared to the non-targeted nanoparticles. The objective is to obtain smart nanoparticles bearing a cleavable masking of the ligands until reaching the tumor cells.Complementarily, there is a need to study different administration routes  as well as the mechanism of clearance, metabolism, and excretion of nanoparticles and their components. Active targeting NCPs containing chemotherapeutic drugs have been demonstrated in various studies, both in vitro and in vivo, to improve selectivity of cellular uptake through receptor-mediated endocytosis. This fact represents several advantages concerning drug efficacy (selective action to cancer cells) and safety (avoid of side effects) over the conventional chemotherapeutic drugs and non-targeted nanoparticles. However, the main limitation for the clinical application is that their use is limited to only certain types of cancer that express specific receptors on the cell surfaces. Selection of the targeting nanoparticles is determined by the types of target proteins or receptors expressed on cancer cell surfaces. Clinical studies are required to confirm their application in cancer patients.NCPs for cancer therapy have evolved enormously during the last decade and it is expected to end up in clinical assays and clinical practice in a short time. Therefore, it is necessary for expert and emergent research groups in areas such as chemistry, biology, medicine, chemical engineering, among others, in order to optimize many of the proposed examples and new ones that may reach clinical application. The control of the physicochemical properties of NCPs and the exploitation of multifunctionality aspects are the main reasons for their increasing applications as anticancer agents. Moreover, there is still a lack of knowledge in the control of biodegradation and metabolic processes, the interaction with the immune system, and the excretion mechanisms. From a technical point of view, there are still some limitations concerning the cost of production and manufacturing of nanoparticle platforms that require sophisticated technology.Since the early 1990s, nanoparticle drug delivery systems have been used in the clinic to improve the delivery of different therapeutics. Over past decades, newer generations of nanoparticles like NCPs have emerged to try to reach clinical trials and be approved for various applications. However, there is still a long way toward the complete regulation of nanomedicines. The need for more sophisticated nanostructured designs also requires a careful understanding of pharmacokinetic and pharmacodynamic properties of nanomedicines, determined by the respective chemical composition and physicochemical properties, which poses additional challenges in regulatory terms. To avoid any concern, it is necessary to establish an unambiguous definition of nanomaterials and work on the regulatory process to facilitate the reach of nanoparticles to the market and the clinical use.


There is lack of information about prevalence of affective and psychotic disorders triggered by traumatic events among people living in war-affected regions. This study is aimed at determining the prevalence rate of affective and psychotic disorders and the associated factors in a war-torn eastern part of Democratic Republic of the Congo. st January 2019 to 31st December 2019 at Cepima and Muyisa health centers. This study enrolled 344 patients that had experienced traumatic events in Eastern Democratic Republic of the Congo from the 1119 participants, of whom 229 had positive bipolar affective disorder and 115 patients had psychotic disorders.  This epidemiological cross-sectional descriptive study was carried out from 1 The results revealed that bipolar affective disorders were two times more than psychotic disorders. Sexual abuse, sudden death of a relative, kidnapping, the physical torture, and childhood trauma were the psychological factors correlated to the occurrence of bipolar affective and psychotic disorders.  It was concluded that the traumatic experiences were precursors for the occurrence of bipolar affective and psychotic spectrum disorders. Affective and psychotic disorders affect both men and women equally with a prevalence rate ranging between 1 and 3% in the general population . BipolarFurther, psychotic features have been linked to depression and anxiety in 9% of the people with history of exposure to stressful life events. In conflict settings, studies have shown that both bipolar affective and psychotic disorders have social, psychological, or physical impairments and affect the quality of life and have been linked to premature death  in additst January 2019 and 31st December 2019. Of these, 352 participants met the fifth edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria for bipolar affective and psychotic disorders [To carry out this epidemiological cross-sectional descriptive study, 1119 patients were screened between 1isorders . Eight pisorders . PatientThe study was approved by the Academic Board of the Catholic University of Graben. Permission to carry out the study was received from the Cepima and Muyisa mental health centers. A valid informed consent was received from the participants with clarity on their mental status and who are able to understand the content of the consent form. The study has been carried out according to the Helsinki Declaration.Two trained research assistants supervised by the first author collected data using a comprehensive questionnaire that consisted of the Mini International Neuropsychiatric Interview (MINI) version 6.0 and Harvard Trauma Questionnaire (HTQ)  as well The collected data were entered into STATA version 13 packages for statistical analysis. The prevalence of affective and psychotic disorders among participants was expressed as proportions of participants with the respective required diagnosis. Logistic regression was performed to assess the correlation between the factors associated with bipolar and psychotic disorders. The measure of association was odds ratios, and the threshold of statistical significance was set at 0.05.p value = 0.002) and psychotic disorder (p = 0.004). Also, having attained primary level of education was positively correlated to affective disorder  and psychotic disorder . Also short duration (6 months) following a traumatic was significantly associated with bipolar affective disorder  and psychotic disorder , sudden death of a loved one , and kidnapping . On the other hand, traumatic events significantly associated with psychotic disorder included physical abuse , sexual abuse , and childhood trauma  (Majority (70.3%) of the participants with bipolar disorder had been exposed to a traumatic event, compared to 46% of patients with psychotic disorders. Traumatic events significantly associated with bipolar affective disorder included sexual abuse  .The present study examined the relationship between the experienced traumatic events and onset of bipolar affective and psychotic disorders at two mental health centers in armed conflict-affected areas in DRC. The findings indicated that factors significantly associated with bipolar affective and psychotic disorders included sexual and physical abuses, childhood trauma, and kidnapping while the prevalence of bipolar affective disorder was higher than that of psychotic disorders among the participants.This study evidenced that experiencing traumatic events in armed conflict-affected areas was followed by the occurrence of affective disorders two times more than the psychotic disorders. This finding is in accordance with the study of Palmieri et al.  who founOur results showed that the gender was associated with the affective disorders and psychotic disorders; especially, the female sex was more represented than the male. Similarly, Belteczki et al.  illustraWe observed that the majority of participants diagnosed with bipolar affective disorder were married, practiced business and farmer jobs, and had attained secondary and university levels compared to those with psychotic disorders. This is in agreement with a number of studies which investigated the rates of bipolar affective disorder according to the sociodemographic variables . There iAdditionally, our findings showed a strong association between the female sex and the early onset and the bipolar affective and psychotic disorders. This is in agreement with results of Charlson et al.  who founThere is also emerging evidence that bipolar affective disorder in the depressive phase was the common bipolar affective disorder among the study participants, while schizoaffective disorder was the most common psychotic disorder detected. These results are in contrast to most previous studies which found a higher prevalence of mania and hypomania , 20. TheIn contrast to other studies, traumatic events were found more among patients with bipolar affective disorder compared to those with psychotic disorders . Our stup value = 0.012) comparing to the sexual abuse, kidnapping, and sudden death of a loved person which were more correlated to the occurrence of psychotic disorders. These results are in contrast with other studies which found a strong correlation between sexual abuse and occurrence of bipolar disorder. The cumulative trauma was more correlated to psychotic disorder than affective disorders by several researchers [Our findings revealed that the physical abuse was significantly associated with bipolar affective disorder (OR: 1.1, 95% CI: 0.39-2.01, earchers , 23, whiearchers . The strearchers , 25 whicearchers . The psyThis study is the first documented study carried out in the war-torn area eastern of the Democratic Republic of the Congo. Its findings are an additional update to the knowledge on major psychiatric disorders following the traumatic events in an armed conflict setting.We could not establish causal-effect relationship given the cross-sectional descriptive study. Also, given that the onset of mental illness is within 6 months, the similarity between mood disorders and PTSD leads to exclude the trauma-related disorders based on clinical features of trauma-related disorders.Traumatic events were significantly associated with affective disorder than psychotic disorders. Bipolar affective disorder depressed phase and the schizoaffective disorder were the common disorders found among the study participants. The findings suggest that physical torture, sexual abuse, childhood trauma, sudden death of a loved one, and kidnapping may be risk factors for occurrence of affective and psychotic disorders among people who have experienced war and conflict. The study findings emphasize the impact of exposure to conflict and onset of the bipolar affective and psychotic disorders.


Objectives: Lymphovascular invasion (LVI) is correlated with unfavorable prognoses in several types of cancers. We aimed to identify the informative features associated with LVI and to determine its prognostic value in colorectal cancer (CRC) patients.Methods: We retrospectively analyzed 1,474 CRC patients admitted in Wuhan Union Hospital between 2013 and 2017 as the development cohort and 549 CRC patients from The Cancer Genome Atlas (TCGA) database as the validation cohort. Logistical and Cox regression analyses were conducted to determine the oncological and prognostic significance of LVI in CRC patients. A survival nomogram based on LVI status was established using the Wuhan Union cohort and validated using TCGA cohort.Results: The LVI detection rates were 21.64% in the Wuhan Union cohort and 35.15% in TCGA cohort. LVI was closely correlated with advanced T stage, N stage, and TNM stage. LVI positivity was an independent biomarker for unfavorable overall survival  and worse disease-free survival  in CRC patients. The survival nomogram incorporating LVI exhibited good predictive performance and reliability in the Wuhan Union cohort and TCGA cohort.Conclusion: LVI is a significant indicator of advanced stage and is remarkably correlated with worse prognosis in CRC patients. The survival nomogram incorporating LVI may assist clinicians to better strategize the therapeutic options for patients with CRC. Colorectal cancer (CRC) is one of the most common types of malignant tumors occurring in the gastrointestinal tract and the main cause of cancer-related death Although a panel of clinical studies have explored the association between LVI and survival outcomes in patients with CRC, the results were inconsistent th edition of the American Joint Committee on Cancer TNM staging system. Patients with histologically diagnosed CRC, patients whose LVI was diagnosed based on a postoperative histopathology report, and CRC patients who underwent surgical treatment were included in the study. By contrast, patients whose CRC was complicated with other malignant tumors and CRC patients with missing critical information were excluded. Moreover, children patients and patients without surgical resection were also excluded from this study. A total of 1,474 CRC patients with intact data were included in this study. Informed consent was obtained from all participants prior to the commencement of the study. The study was reviewed and approved by the ethics Committee of Wuhan Union Hospital (no. 2018-S377).Newly diagnosed patients with confirmed CRC admitted in Wuhan Union Hospital between July 2013 and September 2017 were enrolled in this study For independent validation, TCGA database The clinical features, which were significantly correlated (P<0.05) with the OS of CRC patients, were identified by conducting a multivariable Cox analysis in the development cohort. These characteristics were selected for the construction of an OS nomogram. The predictive discrimination of the OS nomogram was determined using the receiver operating characteristic (ROC) analysis. The calibration ability of the OS nomogram, as reflected by goodness of fit, was measured using a calibration curve. A DFS nomogram was also established in the same manner.All statistical analyses were performed using the SPSS 21.0 and R software (version 3.1.1). The chi-square test was employed to analyze the differences in categorical indexes between LVI+ and LVI- patients. Multivariable logistic regression was utilized to identify the independent variables affecting LVI+. Cox regression analysis was adopted to identify the potent prognostic factors in patients with CRC. ROC analysis was performed to assess the predictive ability of the survival nomogram. The Kaplan-Meier method and log-rank test were used to estimate the prognostic significance of LVI and the nomogram. Two-sided P values of less than 0.05 were considered significant.LVI tumors were identified in 319 (21.64%) of the 1,474 CRC patients with CRC in the Wuhan Union cohort. As listed in Table To determine which clinical variables could independently affect the incidence of LVI, univariable and multivariable logistical regression analyses were performed in the Wuhan Union cohort Table . The mulWe initially analyzed the prognostic value of LVI in the Wuhan Union cohort. As shown in Table A Cox regression model was employed to explore the influence of LVI and other covariates on OS in patients with CRC from Wuhan Union cohort. As shown in Table First, we used TCGA cohort to externally verify the discrimination and calibration of the OS nomogram. As shown in Figure In this study, we initially investigated the oncological significance of LVI in CRC patients who underwent surgical resection and found that LVI was a histologic index of advanced stage in CRC. T4 stage, N3 stage, stage IV, and absence of radiotherapy were independent risk factors for LVI, which were never reported in the previous studies. Then, we further probed the prognostic value of LVI both in the Wuhan Union cohort and TCGA cohort, and the results were almost consistent. Finally, we constructed a survival nomogram based on the LVI status for the risk stratification of CRC patients who underwent surgical treatment in the Wuhan Union cohort. We also validated the survival nomogram using the clinical data from TCGA cohort. Indeed, this multicenter retrospective analysis systemically illustrated the oncological impact of LVI and was the first to establish a survival nomogram incorporating the LVI status for the risk stratification of patients with CRC.The incidence of LVI was not unified in the published studies. Kim et al. Although several studies have already reported the association between the presence of LVI and CRC, most of them investigated this association in patients with a specific stage, such as stage I The College of American Pathologists recommended the assessment of CRC patients for presence of LVI due to its highly important clinical significance Although this is a multicenter research with relatively large sample size, two limitations still exist. One of the limitations was the retrospective nature of the study, and several relevant variables, such as tumor budding and microsatellite instability, were not included in our analysis. Another limitation was that the survival nomogram for CRC patients was not validated in the perspective cohort. Hence, well-designed perspective studies investigating the oncological and prognostic significance of LVI in CRC patients are warranted in the future.Our data showed that LVI may serve as a significant indicator for aggressive tumor behavior and is remarkably correlated with worse prognosis in CRC patients. Moreover, the survival nomogram containing LVI obtained an extremely good predictive ability for predicting OS and DFS in CRC patients, indicating that the survival nomogram can be utilized as a useful prognostic system for individual estimation of prognosis.The study plan was checked and approved prior to the beginning of the research by the ethics Committee of Wuhan Union Hospital (No. 2018-S377). The informed consent was obtained from the all the participants.All data in our study are available from the corresponding author upon reasonable request.


According to the World Health Organization, the prevalence of cancer has increased worldwide. Oncological hyperthermia is a group of methods that overheat the malignant tissues locally or systematically. Nevertheless, hyperthermia is not widely accepted, primarily because of the lack of selectivity for cancer cells and because the temperature-triggered higher blood flow increases the nutrient supply to the tumor, raising the risk of metastases. These problems with classical hyperthermia led to the development of modulated electrohyperthermia (mEHT). The biophysical differences of the cancer cells and their healthy hosts allow for selective energy absorption on the membrane rafts of the plasma membrane of the tumor cells, triggering immunogenic cell death. Currently, this method is used in only 34 countries. The effectiveness of conventional oncotherapies increases when it is applied in combination with mEHT. In silico, in vitro, and in vivo preclinical research studies have all shown the extraordinary ability of mEHT to kill malignant cells. Clinical applications have improved the quality of life and the survival of patients. For these reasons, many other research studies are presently in progress worldwide. Thus, the objective of this review is to highlight the capabilities and advantages of mEHT and provide new hopes for cancer patients worldwide. Cancer is the second leading cause of death worldwide and is considered a universal public health problem with a large impact on healthcare costs. According to the World Health Organization , cancer Ever since cancer was discovered, researchers have struggled to find the best treatment for this lethal disease. As a result, many types of treatments have been developed, including surgery, chemotherapy, radiotherapy, and, more recently, immunotherapy. Among these modalities, the correct choice for patients varies depending on the type and stage of their disease.One method known to ancient medical practitioners was oncological hyperthermia. This method was described as the overheating of malignant tissues, either locally or systematically. It was the first known oncological therapy used by Hippocrates . In modeThis technique is used in five continents and 34 countries, with approximately 400,000 treatments performed each year . HoweverThe mEHT technique is based on two principles: (1) it replaces the single temperature concept that was used previously in hyperthermia with energy that is measured in (kJ/kg), returning the technique to the gold standard dose concepts known to radiation oncologists [4]; (2) it selectively delivers this energy into the tumor without affecting the healthy neighbouring tissue , 8. To s+ and Na+ transport; thus, the membrane potentiation weakens [The ability of cancer cells to produce sufficient ATP is low. A large amount of ATP is needed for proliferative energy consumption. Cancer has less ATP for active membrane stabilization by K weakens The cellular membrane of cancerous cells is electrochemically different from normal cells, since they are negatively charged on average , 18The composition of membrane lipids and sterols differs dramatically between cancer cells and normal healthy cells –21The basic metabolic differences between cancer and noncancer cells make their electrical recognition possible  and incl+, Mg2+, and Ca2+ ions increases, whereas the efflux of Na+ and water transport from the cell decreases. Accordingly, the cell swells, which causes an additional reduction in its membrane potential. Furthermore, as the network of cellular connections (cadherins and junctions) [As a result of these differences, the membrane permeability of cancer cells is altered. Thus, the efflux of Knctions)  are broknctions) , while tnctions) , 23.Using mEHT requires a very simple methodological setup  that depAmong the numerous advantages of using mEHT, the main one is the localisation of the high thermal load to only a narrow and precise region of the plasma membrane of the malignant cell , where tDue to the proper selection and the individual adaptation of the treatment, mEHT is highly personalised . ConsequIn prior studies, phantom measurements of the thermal effects caused by increased temperatures were taken in chopped meat and the liver of a pig . ImportaMany researchers have investigated the effects of mEHT on various in vitro cell cultures and in vivo allografts and xenografts. Comparisons have shown significant differences between cells treated with the same temperature through mEHT and conventional hyperthermia , 57, eve+ and CD8+ that were activated in the group treated with both mEHT and DC immunotherapy. Moreover, this group showed a high level of expression of a different heat shock protein (GP96), which plays an important role in the uptake of antigens by DCs [The mechanism by which mEHT produces apoptosis is important, leading to damage-associated molecular pattern (DAMP) signals that cause immunogenic tumor cell death (ICD). DAMP signals can stimulate the uptake of tumor antigens by antigen-presenting cells . The pros by DCs .+, killing T cells prepared for the immune responses [+. Another explanation may be that the available DCs differentiated into immunosuppressive regulatory forms due to the poor TME and inhibited the activation of T cells, missing a characteristic to block the cancer progression [A systemic tumor-specific immunological response could be a key in successfully treating cancer patients. The tumor microenvironment (TME) triggers the immunological response . The penesponses . This coesponses . The reagression . The seagression . Althouggression , 77–80.A study by Qin et al. that used both DC immunotherapy and mEHT interested the researchers in studying mEHT and its abscopal effect in experimental in vivo models. In addition to the help its ICD production provides in forming APC, another advantage of mEHT is its tumor-selective focus, which does not affect the available immune cells. As a result, they remain intact for the expected immune actions. In one study, a CT26 murine colorectal cancer allograft model was used in a combined DC and mEHT therapy . The resAnother study investigated the molecular mechanism underlying the cytotoxic effects of mEHT on different cell types of hepatocellular carcinoma (Huh7 and HepG2) and found that treating hepatocellular carcinoma cells with mEHT increased the inhibitory effect due to a subset of molecular changes. The molecular changes included the upregulation of septin 4 (SEPT4) and inhibition of G-protein-coupled receptor 64 (GPR64), a key regulator of invasiveness, accompanied by the renewal of cyclin-dependent kinase inhibitor p21. These changes enhanced the apoptotic signalling caused by mEHT. In addition, mEHT inhibited the growth of hepatocellular carcinoma xenograft in nude mice .Recently, a comprehensive review was published showing the clinical achievements of mEHT that were mirrored in numerous publications . Many prThe clinical applications of mEHT fit well with various immuno-oncological therapies, like oncolytic viral treatments –92 and cThe effectiveness of modulated electrohyperthermia as a new hyperthermia method has been shown in numerous studies. Its success derives from nanoscopically heating cancer cells with a high degree of cellular selectivity, which changes the heating paradigm from isothermal homogenous heating to selected cellular heating using the natural heterogeneity of the tumor and its host. In addition, this technique enhances the immune-specific response, which promotes the supporting natural, protective, and defending mechanisms of the human body.  Thus, mEThis technique has minimal toxicity and side effects. Clinical studies show that mEHT improves the quality of life and survival rate of patients. Despite the present results, more in vitro experiments and clinical pieces of evidence have to be collected for a broader range of applications and better results. This treatment could give new hope to cancer patients.


The field of cell therapy and regenerative medicine can hold the promise of restoring normal tissues structure and function. Additionally, the main targets of stem cell-based therapies are chronic diseases and lifelong disabilities without definite cures such as osteoporosis. Osteoporosis as one of the important causes of morbidity in older men and post-menopausal women is characterized by reduced bone quantity or skeletal tissue atrophy that leads to an increased risk of osteoporotic fractures. The common therapeutic methods for osteoporosis only can prevent the loss of bone mass and recover the bone partially. Nevertheless, stem cell-based therapy is considered as a new approach to regenerate the bone tissue. Herein, mesenchymal stem cells as pivotal candidates for regenerative medicine purposes especially bone regeneration are the most common type of cells with anti-inflammatory, immune-privileged potential, and less ethical concerns than other types of stem cells which are investigated in osteoporosis. Based on several findings, the mesenchymal stem cells effectiveness near to a great extent depends on their secretory function. Indeed, they can be involved in the establishment of normal bone remodeling via initiation of specific molecular signaling pathways. Accordingly, the aim herein was to review the effects of stem cell-based therapies in osteoporosis. Osteoporosis as a chronic and long-term skeletal disorder is more common in senile people (in men after age 65 and women after age 55 years) –4. AccorBone as a highly dynamic tissue continuously undergoes modeling and remodeling via activation of bone cells   40–42).–42.40–42As a result of the aging process, reduction in osteoblast number, function, and longevity, lead to bone formation decreasing However, bone resorption is exceeded due to sex hormones defection. Accordingly, individuals are predisposed to osteoporosis and osteoporotic bone fractures –77. In fOsteoporosis cannot be cured but some of the pharmacological and non-pharmacological treatment approaches can manage it  through The clinical demand for new therapeutic methods has been led to progress in stem cell therapy and regenerative medicine , 95. In in vitro and in vivo  (from the inner cell mass of a blastocyst) and induced pluripotent stem cells (iPSCs) (embryonic–like stem cells reprogrammed from adult cells) toward the osteoblasts and osteocyte-like cells (bone-forming cells), some of investigations were shown that application of these most known pluripotent stem cells in osteoporosis treatment is limited due to ethical concerns , 118. ReIn osteoporosis, there is a reduction in endogenous MSCs function . Accordingly, they are the most common types of stem cells investigated in osteoporosis treatment. In this respect, examples of MSCs transplantation in osteoporotic animal models and humans were shown in The burden of osteoporosis is raised by an increase in the proportion of older persons in societies. Routine treatments only alleviate the symptoms partially. Hence, they are not sufficient enough. Therein, regenerative medicine sheds light on the treatment of osteoporosis. Specifically, MSCs therapy is the most common technique of regenerative medicine in osteoporosis treatment. Moreover, using small molecules  which employ endogenous stem cells for osteoporosis treatment will be intertwined in future management , 144. DeBA contributed substantially to the conception and design of the study. MP conducted search strategy and data collection. MS and SA-M drafted critical revision of the article. PG and KG revised the article critically for important intellectual content. NM gave final approval of the version to be submitted and any revised version. BL agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brain metastases are significant causes of morbidity or mortality for patients with metastatic cancer. With the application of novel systematic therapy and improvement of overall survival, the prevalence of brain metastases is increasing. The paradigm of treatment for brain metastases evolved rapidly during the last 30 years due to the development of technology and emergence of novel therapy. Brain metastases used to be regarded as the terminal stage of cancer and left life expectancy to only 1 month. The application of whole brain radiotherapy for patients with brain metastases increased the life expectancy to 4–6 months in the 1980s. Following studies established surgical resection followed by the application of whole brain radiotherapy the standard treatment for patients with single metastasis and good systematic performance. With the development of stereotactic radiosurgery, stereotactic radiosurgery plus whole brain radiotherapy provides an alternative modality with superior neurocognitive protection at the cost of overall survival. In addition, stereotactic radiosurgery combined with whole brain radiotherapy may offer a promising modality for patients with numerous multiple brain metastases who are not eligible for surgical resection. With the advancing understanding of molecular pathway and biological behavior of oncogenesis and tumor metastasis, novel targeted therapy including tyrosine-kinase inhibitors and immunotherapy are applied to brain metastases. Clinical trials had revealed the efficacy of targeted therapy. Furthermore, the combination of targeted therapy and radiotherapy or chemotherapy is the highlight of current investigation. Advancement in this area may further change the treatment paradigm and offer better modality for patients who are not suitable for surgical resection or radiosurgery. Brain metastasis is always an indication of poor prognosis, with short overall survival, progression-free survival, and neurological deterioration . The incThe main goal of treatment of brain metastases is to achieve local control of the metastatic lesion, to improve life quality and to prevent death from neurological disease . The treConvention therapy including surgical resection, whole brain radiotherapy (WBRT), and stereotactic radiosurgery (SRS) and the combination of them were introduced to the treatment of brain metastases since 1980s. The combination of these modalities remains the preferred treatment for selected patients, especially for those with good systematic performance. The advancement in these methods is continuing to extend their application.Surgical resection is indicated for patients with solitude brain metastasis in accessible location or with tumor in large size causing brain edema or hydrocephalus. Patients with controlled disease and good condition are preferred to surgical resection.In the 1980s, several studies revealed benefits of surgical resection for patients with brain metastases . Afterwavs 6 months). There is no difference between the survival of patients with all lesions resected and patients in the additional group.While surgical resection exhibited to be beneficial for patients with one metastasis and good systemic condition, there is not enough robust evidence to evaluate the role of surgical resection for multiple brain metastases . Few retAs regards to the surgical resection of the recurrent brain metastases, previous studies revealed that reoperation resulted in significant function improvement, better quality of life, and protracted overall survival , 17. TheAdvantages of surgical resection include providing histological diagnosis, avoiding long-term use of steroid, immediate amelioration of mass effect, and neurologic deficit . The ratThe life expectancy used to be only 1 month for patients with brain metastases, and the use of steroids protracted the overall survival to 2 months . WBRT isIn the 1970s, the Radiation Therapy Oncology Group (RTOG) conducted a series of clinical trials to examine the efficacy of radiation therapy and explore the appropriate fractionation schemes. Several randomized controlled trials (RCTs) established the WBRT as an important adjunct therapy of surgical resection and SRS, which will be discussed later in this article.The toxicity of WBRT remained the concern of clinical treatment. Brown et al. launched a randomized, double-blinded, placebo-controlled trial to evaluate whether memantine is efficient in palliating the neurological decline . They tuAs regards to the influence of types of tumor, the result of Sundstrom JT’s study showed no significant difference of the overall survival between patients with classic radioresistant tumor and the rest . HoweverIn 2006, the American Association of Neurological Surgeons (AANS) and American Society for Therapeutic Radiology and Oncology (ASTRO) defined stereotactic radiosurgery as “a distinct neurosurgical discipline that utilizes externally generated ionizing radiation to inactivate or eradicate defined targets in the head or spine without the need to make an incision.” SRS is usually performed in a single session, up to a maximum of five sessions, using accelerator under the guidance of real-time imaging. Since its introduction, SRS has been evolving from an investigational concept into one of the mainstream neurosurgical modality for various brain disorders .The SRS was first evaluated for its efficacy by combing with WBRT to compare with WBRT alone. Two randomized clinical trial confirmed its benefit in overall survival and local control for patients with up to four brain metastases , 24. AndWith the confirmation of the benefit of SRS, researches were designed to look into the necessity of WBRT by comparing SRS plus WBRT and WBRT alone for patients with limited number of brain metastases (< 4). In the randomized controlled trial (RCT) conducted by Aoyama et al., 132 patients were assigned to either SRS plus WBRT or SRS alone . While tPrevious studies limit the eligibility of patients as having no more than four brain metastases. With the development of technology and the application of SRS for more lesions in one session, further studies investigated the efficacy of SRS for patients with more lesions. The prospective observational study conducted by Yamomoto et al. revealed that SRS alone without WBRT in patients with five or more brain metastases is non-inferior to that in patients with two to four lesions in terms of overall survival and rate of treatment-related adverse events . The folCombination of conventional therapy including surgery and radiotherapy is the current standard modality for patients with brain metastases, due to the robust evidence showing its efficacy and moderate complication. Several prospective studies as well as systematic review compared the benefit of these modalities and recommended management strategy has been designed for patients with brain metastases , 13, 30.Several clinical trials investigated the superiority of surgical resection plus WBRT against surgery or WBRT alone.vs 6 months) and functionally independent survival. Patients with combined modality also benefited from immediate functional improvement. However, another clinical trial failed to demonstrate the benefit of additional surgery to WBRT. There is no significant difference between the overall survival, mortality, and morbidity rate or cause of death of the patients in two groups.Two prospective randomized trials compared the surgical resection plus WBRT versus WBRT alone , 31. VecA retrospective study conducted by Rades et al.  evaluateIn 1998, the result of a randomized clinical trial established the status of postoperative WBRT as the standard care for patients with brain metastases . A totalAnother three retrospective cohort studies also showed superiority of combined surgical resection and WBRT against surgery alone for patients with brain metastases from lung cancer and melanoma –37.Several retrospective studies compared the efficacy and toxicity of these two modalities , 39. TheHowever, there is no evidence to advocate the use of SRS for patients with a lesion > 3 cm or for those with four or more brain metastases.With the advanced understanding of molecular pathways that mediate oncogenesis and brain metastasis, more preclinical and clinical research investigated the clinical use of targeted therapy for brain metastases . While cThe discovery of epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancer and other types of cancers and the application of EGFR inhibitor have launched a new era of personalized medicine in patients with advanced non-small cell lung carcinoma (NSCLC) . About 2Traditional chemotherapeutic agents failed to penetrate the BBB due to its large molecular weight. Similarly, the cerebrospinal fluid (CSF) penetration of the first- and second-generation TKI is low, with average CSF penetration rates of < 1%, 1%–3%, and 3%–6% reported for afatinib, gefitinib, and erlotinib, respectively. However, the penetration of gefitinib is enhanced in patients with brain metastases, possibly due to the tumor-induced BBB disruption .Nevertheless, EGFR TKIs has demonstrated efficacy for patients with brain metastases. A meta-analysis revealed that EGFR TKIs combined with SRS or WBRT lead to improved response rate, prolonged progression-free survival (PFS), and overall survival . The resThe major challenge TKIs encountered is acquired drug resistance, which occurs in a majority of patients treated with the first and second generation of TKIs . A new gCTLA-4 is a protein receptor functioning as a check point in the T cells to downregulate its immune response when bounded to CD80 or CD86. It is commonly expressed in the regulatory T cells and upregulated in the activated conventional T cells, which is a notable phenomenon in cancers . IpilimuAn open-label, phase 2 trial investigates the efficacy and safety of ipilimumab alone in patients with melanoma and brain metastases. The central nervous system (CNS) response rate is 16% in asymptomatic patients and 5% in symptomatic patients. The overall survival is 2.7 months for asymptomatic patients and only 1.3 months for symptomatic patients.In an open-label, single-arm phase 2 trial, 86 eligible patients were treated with fotemustine plus ipilimumab . The disProgrammed cell death protein 1 (PD-1) is another important check point expressed in the T cells that negatively regulate the immune response when bounded to its ligands programmed death-ligand 1 (PD-L1) or programmed death-ligand 2 (PD-L2). It works by promoting the apoptosis of antigen-specific T cells and suppressing the apoptosis of the regulatory T cells , 52.Approved PD-1 inhibitors include nivolumab and pembrolizumab, while approved PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab. Several clinical trials are ongoing to evaluate their efficacy and safety on melanoma, NSCLS, renal cell carcinoma (RCC), and so on.A phase II clinical trial investigated pembrolizumab for patients with melanoma of NSCLC and untreated brain metastases (NCT02085070) . A totalAn open-label, multicenter, phase study investigated the efficacy and safety of combined nivolumab and ipilimumab for patients with brain metastases . Ninety-The rationale behind the combination of the immunotherapy was derived from abscopal effect. Abscopal effect refers to the phenomenon that treatment of the local tumor leads to the concurrent shrinkage of the metastatic tumor at distant sites. The hypothesis is that radiation leads to the death of tumor and thus the liberation of tumor-associated antigens (TAAs) . These aCombining radiotherapy with immunotherapy provides opportunities for boosting abscopal response rate and further use of radiotherapy for local and metastatic tumors .Several studies had been conducted to investigate the efficacy and safety of the combination of immunotherapy and radiotherapy for brain metastases.One study proved that the concurrent use of immunotherapy and SRS resulted in appreciable reduction in lesion volume . A totalHowever, it should be noted that another retrospective study showed no significant difference between the survival of patients who underwent SRS alone and patients with concurrent ipilimumab and SRS .A phase 1 study was designed to determine the safe dose of ipilimumab with SRS or WBRT for the treatment of melanoma with brain metastasis . The medSeveral trials are ongoing to investigate the efficacy, safety, proper timing of combination of radiotherapy, and immunotherapy . The resThe paradigm changed rapidly in the treatment of brain metastases. The selection of modality is of vital importance for the benefit of the patients. Surgical resection plus WBRT is preferred for patients with single lesion and good condition. Surgery can also lead to immediate remission of the clinical neurosurgical symptoms. SRS plus WBRT provided better neurological protection despite the shortening of overall survival. Special WBRT or adjunct therapy can be applied to decrease the rate of neurocognitive decline. The use of novel targeted therapy may put forward a new method for the patients with brain metastases, especially those with numerous multiple brain metastases. The type of primary tumor and the mutation should guide the use. Further prospective clinical trials are needed to evaluate the efficacy and safety.