Pancreatic ductal adenocarcinoma (PDAC) patients display distinct phenotypes of cachexia development, with either adipose tissue loss preceding skeletal muscle wasting or loss of only adipose tissue. Activin A levels were measured in serum and analyzed in tumor specimens of both a cohort of Stage IV PDAC patients and the genetically engineered KPC mouse model. Our data revealed that serum activin A levels were significantly elevated in Stage IV PDAC patients in comparison to age-matched non-cancer patients. Little is known about the role of activin A in adipose tissue wasting in the setting of PDAC cancer cachexia. We established a correlation between elevated activin A and remodeling of visceral adipose tissue. Atrophy and fibrosis of visceral adipose tissue was examined in omental adipose tissue of Stage IV PDAC patients and gonadal adipose tissue of an orthotopic mouse model of PDAC. Remarkably, white visceral adipose tissue from both PDAC patients and mice exhibited decreased adipocyte diameter and increased fibrotic deposition. Strikingly, expression of thermogenic marker UCP1 in visceral adipose tissues of PDAC patients and mice remained unchanged. Thus, we propose that activin A signaling could be relevant to the acceleration of visceral adipose tissue wasting in PDAC-associated cachexia. Unlike starvation where adipose tissue mass is lost while skeletal muscle mass is preserved, cachexia patients lose weight from both compartments3. The current international consensus for diagnostic criterion for cancer cachexia is weight loss > 5% over 6 months; or BMI < 20 and any degree of weight loss > 2%; or appendicular skeletal muscle index consistent with sarcopenia and any degree of weight loss > 2%4. Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer death in the United States and is projected to become the second leading cause by 20305. Due to lack of early clinical signs, more than 85% of patients present with advanced metastatic disease by time of diagnosis6. Prognosis for patients with metastatic disease remains dismal, with a 5 year survival rate of only 2% and median survival time of 3–6 months7. A major contributor to the low survival rate is cachexia, a frequent and prominent feature of pancreatic cancer. Notably, 80% of pancreatic cancer patients exhibit symptoms of cachexia at later stages of disease development8. Median weight loss close to time of death has been reported to correspond to 25% of pre-illness weight9.Cancer cachexia is a devastating, multifactorial, and often irreversible syndrome that accounts for up to 20% of cancer deaths10. One recent report demonstrates that subcutaneous adipose tissue loss is initiated in patients prior to loss of skeletal muscle and PDAC diagnosis, identifying three distinct and progressive phases of metabolic and soft tissue changes in pre-diagnostic PDAC starting up to 18 months before diagnosis11. This phenomenon insinuates that a causal factor for cachexia has already begun to exert systemic effects prior to presentation of prominent cachectic symptoms.Adipose tissue serves as a metabolically dynamic endocrine and energy-storage organ of the body and is primarily composed of adipocytes. In the current research paradigm, most studies and clinical trials have focused on skeletal muscle atrophy rather than adipose tissue loss in cancer cachexia. However, emerging reports indicate that PDAC patients display distinct phenotypes of cachexia development, with either adipose tissue loss preceding skeletal muscle wasting or loss of only adipose tissue13. This phenotypic switch has been shown to take place at the initial stages preceding skeletal muscle atrophy15. However, factors that mediate adipose tissue loss in PDAC patients and their contribution to WAT browning in PDAC-mediated cachexia remain to be unveiled.Brown adipocytes may appear after thermogenic stimuli in WAT in a process known as “browning”. A growing body of evidence from animal studies suggests that WAT undergoes “browning” during cancer cachexia, resulting in increased lipid mobilization and energy expenditure16. The activin signaling pathway has been researched extensively regarding its functions in pancreatic cancer development and progression17. Activin signaling can be exploited by cancer cells for their growth advantage paradoxically18, despite its evident role in tumor suppression19. Multiple studies have implicated a significant role of activin A in skeletal muscle wasting in cancer cachexia and led to propose serum activin A as a prognostic biomarker for cancer patients22. Surprisingly, few studies have been published regarding activin A’s role in adipose tissue loss in cancer cachexia. One study has been published reporting that the combined administration of activin A and interleukin-6 (IL-6) adenovirus vectors resulted in atrophy of WAT in an animal model23, suggesting an interplay between those two factors. A clinical study demonstrated that cachectic patients with gastrointestinal cancer show morphological rearrangement in subcutaneous adipose tissue, resulting in adipose tissue atrophy, formation of fibrotic areas, and immune cell infiltration24. Another study that examined the modulation of extracellular matrix (ECM) components in fibrotic subcutaneous adipose tissue obtained from cachectic gastrointestinal cancer patients showed morphological changes in cancer cachexia concomitant with augmented expression of ECM elements through enhanced signaling of the TGFβ pathway25. While subcutaneous adipose tissue has traditionally been associated with plasma changes that may function as markers of cancer cachexia26, additional studies in pancreatic cancer patients suggested that the rate of visceral adipose tissue loss may be an important indicator of survival. Patients with at least two abdominal CT scans between diagnosis and death, receiving surgery or chemotherapy during cancer progression, were selected. The rate of change (%change/100 days) for subcutaneous adipose tissue was similar to visceral adipose tissue but a change in visceral adipose tissue was significantly correlated with survival in cancer patients27.Activins and inhibins are family members of the TGFβ superfamily involved in a wide range of cellular processes. Activin A is expressed in numerous tissues and plays pivotal roles in regulation of tissue homeostasis, organ development, inflammation, and cell proliferationHere, we investigated the correlation between activin A signaling and visceral adipose tissue remodeling specifically in cachectic PDAC patients, opening a new avenue for finding therapeutic targets to prevent or attenuate the progression of cancer cachexia in patients with pancreatic cancer.All methods were performed in accordance with the relevant guidelines and regulations.n = 34) were obtained through the Rapid Autopsy Program (RAP) for Pancreas in compliance with IRB 091-01. To ensure specimen quality, organs were harvested within three hours postmortem and specimens flash-frozen in liquid nitrogen or fixed in formalin. Informed consent was obtained from all subjects and/or their legal guardian(s). Any studies using tissue solely obtained from the RAP are not considered human subject research because the individuals are not living when the tissue is obtained (per the regulations at 45 CFR 46.102) and therefore would not need IRB approval. The L3 lumbar vertebra landmark was used to estimate fat mass in decedents who underwent CT imaging at time of diagnosis.Human pancreatic specimens and omental adipose tissue from decedents diagnosed with PDAC (n = 25) were obtained from the Tissue Procurement and Storage Facility though the Biorepository Laboratory of the Nebraska Biobank. Frozen visceral adipose tissue from male non-cancer patients were procured from the Research Tissue Bank (n = 7). The banking of excess discarded de-identified human biological materials obtained solely for clinical purposes does not constitute human subject research subject to 45 CFR 46.Serum samples from age-matched, non-cancer patients with no history of endocrine disorders such as diabetes mellitus . Temperature, humidity, and photoperiod (10/14 photoperiod) were kept constant.G12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) was bred by the lab of Dr. Michael A. Hollingsworth as previously described (n = 5)28. We were granted permission for usage of tumor-derived cell lines KPC8060 and KPC8069 that were generated as previously described29. Age-matched healthy C57BL/6 J mice (n = 5) were utilized as controls.A colony of the transgenic mouse model LSL-Kras6 S2-013 cells)30 was orthotopically implanted into the pancreas of adult male and female athymic nude mice (n = 7) by the laboratory of Dr. Pankaj Singh. Age-matched mice who underwent sham operations were utilized as controls (n = 7).A cloned sub-line of human PDAC cell line SUIT-2 . Concentrations for interleukin-6 (IL-6) were determined using the Human IL-6 High Sensitivity ELISA kit , and Mouse IL-6 ELISA kit . All experiments were performed in duplicates.31. Human PDAC cell lines BxPC3 , PANC-1 , and MIA PaCa-2 ; and human cervical adenocarcinoma cell line HeLa  were maintained in RPMI-1640  or Dulbecco’s Modified Eagle Medium  with 10% FBS  and 1% penicillin–streptomycin-glutamine (PSG) . An additional 2.5% horse serum  was added to the culture medium of MIA PaCa-2 cells. The KPC8060 and KPC8069 cell lines were maintained in RPMI-1640 with 10% FBS and 1% PSG. All cells were maintained in a 5% CO2-humidified atmosphere at 37 °C.hTERT-HPNE, a normal cell line of intermediary cells formed during acinar-to-ductal metaplasia, was established and granted permission for usage by Dr. Michel M. OuelletteTo prepare conditioned medium (CM), fresh culture medium was added to confluent cells and maintained for 24 h prior to collection.ex-vivo culture. Briefly, two gonadal and subcutaneous fat pads from one mouse were divided into four pieces by cutting vertically, and each piece was incubated in either RPMI-1640 (control), CM from KPC8069 cells or CM from KPC8069 cells supplemented with the activin-binding protein follistatin 288  for 48 h. The tissues were then fixed in Modified Davidson’s Fixative  and sectioned for Picro-Sirius Red Staining.Gonadal and subcutaneous adipose tissue were obtained from four C57BL/6 male mice aged between 9 and 11 weeks and used for the Total RNA was collected using the RNeasy Plus Mini Kit  or the RNeasy Lipid Tissue Mini Kit . One microgram of total RNA from each of the samples were then converted to cDNA using the High-Capacity RNA-to-cDNA™ Kit . TaqMan® probe-based  or SYBR® Green based  real-time PCR was performed using primer sequences listed in Supplemental Table 4 cells from each of the human pancreatic cancer and normal cell lines were seeded on cover glasses overnight and fixed with 2% formaldehyde. Antigen retrieval was carried out in 10 mM sodium citrate buffer. Samples were then blocked with normal goat serum blocking solution  and treated with primary antibodies .TUNEL was performed using the DeadEnd™ Fluorometric TUNEL System .All images were taken with the EVOS M7000 Imaging System .Samples were homogenized in RIPA buffer  combined with cOmplete™ Protease Inhibitor Cocktail  and PhosSTOP™ . Equal amounts of protein were loaded onto a 4–20% polyacrylamide gel  and transferred to a nitrocellulose or PVDF membrane. The primary and secondary antibodies used are described in Supplemental Table www.graphpad.com). Values of p < 0.05 were considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant.Data were presented by mean ± S.E.M. Serum activin A and IL-6 levels were compared between groups using a nonparametric Wilcoxon rank-sum test due to the departure from the normality. Pearson correlation was used to determine the correlation between activin A and IL-6 levels and activin A levels with metastasis sites and body mass index for patients with PDAC. All figures with statistical analyses were generated using PRISM version 9.2.0  expression and the prevalence of cancer cachexia in 18 types of cancer using RNA-Seq data from The Cancer Genome Atlas (TCGA). Notably, the expression of INHBA was greater in PDAC tumor tissue in comparison to that in matched normal tissue  and CK17 appear to be highly expressed in PDAC tumor tissues in comparison to non-cancerous pancreatic tissues. A large portion of βA-positive cells were found to overlap with CK17 signal  in PDAC tumor specimens and non-cancerous pancreatic tissues. We also investigated the expression of keratin 17 (CK17), a marker of the most lethal molecular subtype of pancreatic cancern = 34) and from healthy donors (n = 25). Serum activin A concentrations of PDAC donors had an average of 5.85 ng/mL, while serum activin A concentrations of healthy donors had an average of 0.91 ng/ml  was greatly reduced   Fig. B. To addas) Fig. C, D. TheUnexpectedly, we were unable to detect an increase in classical brown adipose tissue marker uncoupling protein 1 (UCP1) protein expression per adipocyte in S2-013 mPDAC samples from the gonadal fat pad Fig. E, F. To UCP1 in visceral adipose tissue revealed no significant change with increasing circulating activin A levels in PDAC patients and no significant change between PDAC patients and non-cancer patients  is a notoriously lethal disease with indications that both adipose tissue and skeletal muscle wasting occurs early in disease progression37. The deletion of inhibin elevated circulating activin A by 20-fold and manifested symptoms akin to human cancer cachexia38. A clinical trial in patients with cancer of the lung or the pancreas demonstrated that blockade of ACTRIIB using monoclonal antibody bimagrumab (BYM338) exerts an effect on preserving body weight and baseline of thigh muscle volume, total lean body mass, bone mineral density, and physical activity levels (NCT01433263). While BYM388 appears promising for recovery of skeletal muscle mass, there is no published clinical data regarding the effects of BYM338 on fat mass in patients with pancreatic cancer. To our knowledge, our study is the first to reveal the dose-dependent association between serum activin A levels and loss of visceral adipose tissue in PDAC patients.The first animal study regarding activin A and cancer cachexia was in the inhibin-deficient mouse modelKRAS or p53 mutation status and the production of activin A for promotion of cancer growth (data not shown). Intriguingly, the maintenance of high levels of activin A secretion in KPC8069 cells after changing culture medium implies that there is continuous production of activin A from tumor cells with both KRAS and p53 mutations.The recently published report by Zhong et al. investigates human PDAC tumor expression of activins and related factors in relation to their effects on skeletal muscle loss. Our data concur with conclusions from their report regarding the choreography of systemic effects of activin A by PDAC tumors for the induction of cachectic pathogenesis in patients. The imbalance in activin A expression between PanIN lesions and tumor cells lends credence to a correlation between secretion of activin A and acceleration of cachexia in the disease state. The lack of change in activin A secretion between BxPC3 and PANC-1 cells suggests there may not be a relationship between 25. These shed new light on activation of SMAD-independent signaling pathways that has been described in non-adipose tissue41. Previous reports have posited that targeting of molecules downstream of activin A such as Twist1 or JNK ameliorates cancer-induced muscle cachexia and may prolong survival in animal models of PDAC43. In consideration of our findings, the activity of these molecules may also be targetable in adipose tissue. Given that we showed that browning of white adipose tissue is not the primary mechanism of cachectic fat loss in PDAC patients and mice, activin A seems to mediate an alternative mechanism for triglyceride degradation in visceral adipose tissue. Although our study design is limited to determine the effects of secreted activin A from PDAC tumor cells on visceral adipose tissue, further investigation is currently ongoing to examine the specificities of activin A signaling and the mechanisms by which activin A mediates cachectic fat degradation in PDAC. In summary, our study reveals a novel role of activin A in relation to the loss of visceral adipose tissue in the cachectic state in pancreatic cancer and may open new possibilities for the development of targeted therapies for the treatment of cachexia.Unlike phenomena observed in other studies, visceral WAT in PDAC patients and our mouse model displayed progressive loss of adipocyte function and size without undergoing the browning process. Our results instead demonstrate fibrotic remodeling of human and mouse visceral adipose tissue with elevated serum activin A, suggesting that activin A could induce trans-differentiation of white adipocytes into fibrotic cells. This is parallel to the implication of TGFβ signaling with fibrosis of subcutaneous fat in patients with cancer cachexiaSupplementary Information.


In this paper, we present a novel approach to direction-of-arrival (DoA) estimation using two-row electronically steerable parasitic array radiator (ESPAR) antenna which has 12 passive elements and allows for elevation and azimuth beam switching using a simple microcontroller, relying solely on received signal strength (RSS) values measured at the antenna output port. To this end, we thoroughly investigate all 18 available 3D antenna radiation patterns of the antenna measured in an anechoic chamber with respect to radiation coverage in the horizontal and vertical direction and propose a generalization of the power-pattern cross-correlation (PPCC) algorithm involving a high number of multiple calibration planes (MCP) as well as specific combinations of radiation pattern sets. Additionally, a new way of RSS-based DoA estimation accuracy assessment, which involves thorough testing conducted along the elevation direction when RF signals impinging on the antenna arrive from arbitrary θ angles, has been reported in this paper to verify the overall algorithm’s performance. The results obtained for different signal-to-noise ratio (SNR) levels indicate that two-row ESPAR antenna can produce, even for low SNR values, accurate DoA estimation in the horizontal plane without prior knowledge about the elevation direction of the unknown RF signals by using appropriate combinations of only 12 3D antenna radiation patterns. Wireless sensor networks (WSNs), especially in the Internet of Things (IoT) applications, depend on low-cost wireless transceivers, which are usually integrated with simple microcontrollers to provide the functionality required by different IoT applications ,2,3. To Electronically steerable parasitic array radiator (ESPAR) antenna is one of the promising SBA designs that can successfully be integrated with a WSN node  or a WSNTo further adapt the ESPAR antenna proposed in  to the aOne of the main benefits of ESPAR antennas is that, according to existing literature, they can easily be integrated with a WSN node and provide DoA estimation functionality to the node relying solely on RSS measurements gathered for 12 ESPAR antenna radiation patterns . The PPCTo address WSN connectivity challenges in practical industrial installations ,26 and aCombined connectivity and DoA estimation capabilities can highly improve IoT systems involving WSN nodes and gateways installed on the ground in smart factories, buildings and cities ,6,25,26.In-depth analysis of the antenna from the connectivity perspective with respect to possible beam steering in horizontal and elevation directions;Presentation of an approach for DoA estimation relying solely on RSS values gathered at the antenna output, which is a prerequisite for energy-efficient WSN nodes having DoA functionality ,10, thatProposal of a detailed DoA algorithm performance testing method for more accurate DoA estimation accuracy assessment that involves all θ angles to address strong error variation at low θ angles.Connectivity performance and RSS-based DoA estimation approaches, which are crucial to develop new WSN gateways and nodes integrated with two-row ESPAR antenna, have not been investigated in the original publication . AccordiThe rest of this paper is organized as follows: in sth passive element: The proposed antenna concept has been discussed in  and is pribed in .The fabricated antenna is presented in The main goal of the proposed antenna was oriented towards connectivity improvement by increased angular coverage of the directional radiation pattern that can be switched in both, azimuth and elevation. For this reason, a more detailed analysis of the antenna radiation capabilities has to be considered. In To assess the angular coverage of the antenna that include all possible beam configurations, aggregated radiation patterns need to be considered. This form of presentation is known in the context of time-modulated arrays and 3D coverage analysis for 5G applications where the aggregated characteristic consists of all available antenna radiation patterns to show the complete spatial coverage providable by the antenna . Aggrega be used . In Figuθ = 90° direction  with 2–4° precision being the maximum DoA estimation error T ,28. In cθ angles ,24,28,34The vector form of the PPCC algorithms in Equations (2) and (3) is appropriate for implementation in a WSN node’s microcontroller to compute DoA estimation based on RSS measured for incoming packets. It is also straightforward to use such a node with ESPAR antennas having different number of directional radiation patterns or other switched beam antennas . HoweverTo perform RSS-based DoA estimation based on radiation patterns available for two-row ESPAR antenna, one has to create a generalized PPCC-MCP algorithm that can easily be implemented within simple WSN nodes and involves all possible radiation patterns. Due to their spatial characteristics, it is possible to increase the overall accuracy of the estimation while mitigating the necessity of correct placement of calibration planes. As the two-row ESPAR antenna radiation patterns, which are created using associated steering vectors θ angles.Total number of radiation patterns used in the DoA estimation and H = 4.1 m on a precise and equipped with a digital encoder turntable, which is able to set any 2D position with H = 4.1 m, 8 m from the two-row ESPAR antenna. Every radiation pattern was measured with the angular step I = 360 calibration points were produced for every elevation plane and M = 90 calibration planes were created, namely To verify DoA estimation performance of the two-row ESPAR antenna using the generalized PPCC-MCP method 18 available antenna radiation patterns were measured in our 11.9 m × 5.6 m × 6.0 m anechoic chamber, shown in To examine DoA estimation accuracy, measured calibration planes were imported to Matlab, where 10 snapshots of sinusoidal test signal were generated for all considered test directions s output ,20,23,33M = 9 calibration planes are used. As it was overlooked in [To compare the results with those already available in the literature, the test signal directions have to be set with discrete angular steps equal to ectively ,28,33. Ierved in  that wheN = 18 available antenna radiation patterns were divided into 3 separate groups having different angle tilt in elevation, as shown in To verify DoA estimation performance of the two-row ESPAR antenna using the generalized PPCC-MCP algorithm, which uses all 90 available calibration planes, To test DoA estimation accuracy in a detailed way, 360 test horizontal directions As it can easily be observed in To verify the performance of generalized PPCC-MCP algorithm for different SNR levels, two additional DoA tests, with SNR = 5 dB and SNR = 0 dB, were performed. As it can easily be noticed in In this paper, we show how a two-row ESPAR antenna can be used for RSS-based DoA estimation. To this end, we have presented an analysis of all 18 available 3D antenna radiation patterns, being measured in an anechoic chamber, with respect to radiation coverage in the horizontal and vertical direction as well as their directional performance important in the estimation process. Moreover, we have proposed PPCC-MCP algorithm generalization that involves the usage of a very high number of calibration planes and can handle specific combinations of two-row ESPAR antenna radiation pattern sets. Detailed DoA estimation accuracy assessment, which involves thorough testing conducted along the elevation direction when RF signals impinging on the antenna arrive from arbitrary θ angles, shows that accurate results can be produced for only 12 steering vectors used. Precision lower than 11°, 17°, and 42° have been obtained for all θ angles when SNR was equal to 10 dB, 5 dB, and 0 dB, respectively. These results indicate that a two-row ESPAR antenna can produce accurate DoA estimation in the horizontal plane without prior knowledge about the elevation direction of the unknown RF signals even for low SNR values. Therefore, as the antenna beam switching in elevation and azimuth can be realized using a simple microcontroller and the DoA estimation relies solely on RSS values measured at the antenna output port, the antenna can easily be integrated with a WSN node or a WSN gateway. Moreover, 2D beamforming capabilities and accurate DoA estimation opens up new frontiers for ESPAR antenna applications involving new portable low-cost ground penetrating radars  and noni


The blaNDM gene is globally distributed across a variety of Gram-negative bacteria on multiple plasmids, typically located within highly recombining and transposon-rich genomic regions, which leads to the dynamics underlying the global dissemination of blaNDM to remain poorly resolved. Here, we compile a dataset of over 6000 bacterial genomes harbouring the blaNDM gene, including 104 newly generated PacBio hybrid assemblies from clinical and livestock-associated isolates across China. We develop a computational approach to track structural variants surrounding blaNDM, which allows us to identify prevalent genomic contexts, mobile genetic elements, and likely events in the gene’s global spread. We estimate that blaNDM emerged on a Tn125 transposon before 1985, but only reached global prevalence around a decade after its first recorded observation in 2005. The Tn125 transposon seems to have played an important role in early plasmid-mediated jumps of blaNDM, but was overtaken in recent years by other elements including IS26-flanked pseudo-composite transposons and Tn3000. We found a strong association between blaNDM-carrying plasmid backbones and the sampling location of isolates. This observation suggests that the global dissemination of the blaNDM gene was primarily driven by successive between-plasmid transposon jumps, with far more restricted subsequent plasmid exchange, possibly due to adaptation of plasmids to their specific bacterial hosts.The mobile resistance gene  blaNDM, conferring resistance to carbapenem antibiotics, is globally distributed across Gram-negative bacteria on multiple plasmids. Here, Acman et al. study the dynamics underlying blaNDM dissemination across over 6000 bacterial genomes, and identify mobile genetic elements and specific mobilisation events likely involved in the gene’s global spread.Gene  These antibiotics are used as a first-line treatment for severe infections and to treat multidrug-resistant Gram-negative bacterial infections. As such, the global prevalence of bacteria carrying blaNDM represents a major public health concern.Antimicrobial resistance (AMR) poses a major challenge to human and veterinary health. AMR can be conferred by vertically inherited point mutations or via the acquisition of horizontally transmitted ‘accessory’ genes, often located in transposons and plasmids. The blaNDM was first described in 2008 from a Klebsiella pneumoniae isolated from a urinary tract infection in a Swedish patient returning from New Delhi, India2. While blaNDM now has a worldwide distribution, most of the earliest cases have been linked to the Indian subcontinent, leading to this region being suggested as the likely location for the initial mobilization event6. NDM-positive Acinetobacter baumannii isolates have been retrospectively identified from an Indian hospital in 20057, which remain the earliest observations to date. Nevertheless, an NDM-positive A. pittii isolate was also collected in 2006 from a Turkish patient with no history of travel outside Turkey8.blaNDM on multiple non-conjugative, but potentially mobilizable plasmid backbones7. In addition, blaNDM in these early isolates was positioned within a complete Tn125 transposon with IS26 insertion sequences (ISs) as well as ISCR27 (IS-containing common region 27), suggesting the possibility of complex patterns of mobility since the gene’s initial integration. Subsequent NDM-positive isolates across multiple species consistently harbour either a complete or fragmented ISAba125 (an IS constituting Tn125), immediately upstream of blaNDM, which provides a promoter region10. The presence of ISAba125 in some form in all NDM-positive isolates to date and the early observations in A. baumannii have led to Tn125 being proposed as the ancestral transposon responsible for the mobilization of blaNDM, and A. baumannii as the ancestral host11.Although no complete genome sequences are publicly available from these earliest observations, the first NDM-positive isolates from 2005 were shown to carry 11, with the first six amino acids in NDM matching to those in aphA6, a gene providing aminoglycoside resistance and also flanked by ISAba125. It is hypothesized that ISCR27, which uses a rolling-circle (RC) transposition mechanism13, initially mobilized a progenitor of blaNDM in Xanthomonas sp. and placed it downstream of ISAba12515. At least 29 distinct sequence variants of the NDM enzyme have been described to date16. The most prevalent of these variants is the first to have been characterized, denoted NDM-117. Different NDM variants are mostly distinguished by a single amino-acid substitution, apart from NDM-18 that carries a tandem repeat of five amino acids. None of the observed substitutions occur in the active site and their functional impact remains under debate1.The NDM enzyme itself is of possible chimeric originblaNDM is found in at least 11 bacterial families and NDM-positive isolates have heterogeneous clonal backgrounds, supporting multiple independent acquisitions of blaNDM1. Although blaNDM has been observed on bacterial chromosomes19 it is most commonly found on plasmids, comprising multiple different backbones or types. Thus far, blaNDM has been associated with at least 20 different plasmid types, predominantly IncFIB, IncFII, IncA/C (IncC), IncX3, IncH, and IncL/M, and also in untyped plasmids23. Furthermore, even within the same plasmid type, blaNDM can be found in a variety of genomic contexts, often interspersed by multiple ISs and composite transposons11. The immediate environment of blaNDM has been reported to vary even in isolates from the same patient22. Many mobile elements are thought to play important roles in dissemination, including ISAba125, IS3000, IS26, IS5, ISCR1, Tn3, Tn125, and Tn300026. It is therefore clear that the spread of blaNDM was, and is, a multi-layer process involving multiple mobile genetic elements—‘the mobilome’. blaNDM mobility involves diverse processes, including genetic recombination28, transposition, conjugation and transformation of plasmids29, transduction30, and transfer through outer-membrane vesicles32.blaNDM-positive genomes have led to a better understanding of its evolution1. However, a major difficulty, as for other AMR genes, is relating the diverse genomic contexts to temporal evolution. Here, we outline an alignment-based method to identify flanking structural variants and use it to build a history of the insertion and mobilization events. We compile a global dataset of more than 6000 NDM-positive isolates. In line with previous studies, we identify Tn125, IS26 and Tn3000 as the main contributors to blaNDM mobility but go further and estimate the timing of the initial emergence of blaNDM to pre-1990, around two decades prior to its first detection and rapid dissemination. Our findings suggest that this global spread was driven primarily by transposons, with plasmids playing more of a role in local transmission.Previous surveys of blaNDM , NCBI GenBank34 (n = 1158) and Enterobase35 (n = 1379); bacterial genomes assembled using short-read de novo assembly from NCBI’s Sequence Read Archive (SRA) (n = 882); and newly generated bacterial genomes isolated from 79 hospitalized patients across China and 25 livestock farms assembled using hybrid PacBio-Illumina de novo assembly (n = 104)  carrying at least one copy of . 33 n = 632, NCBIblaNDM-positive isolates from 88 states , European countries (941), USA (461), Thailand (419) and India (361). At least 27 bacterial genera were represented, with a large fraction of Klebsiella and Escherichia isolates  increasing in prevalence after 2012  grouped contigs of the same replicon type and contained a small number of contigs (only 27 clusters included >10 contigs), in line with a diverse and dynamic population of plasmid backbones for blaNDM.We identified 33 different replicon types on 1222 contigs using PlasmidFinder42 Fig. . The mosblaNDM-carrying contigs were associated with small putative plasmids  are inherited by descent and are a result of transposon-mediated plasmid fusion43.The majority  of blaNDM and gain a detailed overview of the possible events in its evolution, we developed an alignment-based approach to progressively resolve genomic variation moving upstream or downstream from the gene  that share a BLAST hit with a minimum length as given by the ‘splitting threshold’ is assessed . This transposition hotspot probably contributes to fragmented assemblies: 2269 contigs were excluded from further analysis for being too short , trpF (6038), dsbD (5551), cutA (2731), groS (2175), groL (1631). When restricted to blaNDM-positive contigs of sufficient length to possibly harbour the full repertoire of these genes (n = 3786), almost half carry all of them . In addition, we find dominant structural variants associated with various source databases and sequence lengths hence diminishing the impact of the sampling bias , cutA 231, groS blaNDM. The putative ancestral Tn125 background, with an uninterrupted downstream ISAba125 element, was seen in contigs mainly from Acinetobacter and Klebsiella  to the blaNDM start codon. These account for a minority  of isolates when sufficiently long contigs are considered. This supports the initial dissemination of blaNDM by Tn125 to other plasmid backbones predominately being mediated by Acinetobacter and Klebsiella, after which the transposon was disrupted by other rearrangements.While we did not observe any strong overall signal in the distribution of associated plasmid backbones, bacterial genera, or sampling locations, closer examination of mobilome features common to sufficiently large numbers of isolates indicated early events in the spread of lla Fig.  top. Con3000, both upstream and downstream, was almost exclusively associated with samples from Klebsiella  was ISCR1 (IS91 family transposase) Fig.  always aNDM Fig. 13. NevergroL gene at high prevalence  is located 236 bp downstream of the ISCR27 transposase stop codon. The region downstream of this stop codon in all structural variants bearing a complete ISCR27 is highly conserved for at least 750 bp  that mapped to the downstream end of the contigs were screened against the ISFinder database47. The ≥50 bp overhangs associated with 3000–3300 long flanks downstream of blaNDM corresponding to the largest observed drop almost exclusively match the left inverted repeat of the IS26 sequence .Three sharp drops in the number of considered contigs at particular distances downstream of 26, although often found in two adjacent copies forming a seemingly composite transposon, is a so-called pseudo-composite (or pseudo-compound) transposon48. In contrast to composite transposons, a fraction of DNA flanked by the two IS26 is mobilized either via cointegrate formation or in the form of a circular translocatable unit (TU), which consists of a single IS26 element and a mobilized fraction of DNA, and inserts preferentially next to another IS2649. Taken together, the presented results, including Supplementary Fig. blaNDM carrying contigs in the dataset: (i) the presence of IS26 TUs in the host cell; (ii) other circular DNA formations mediated by plasmid recombination, transposons43 or ISCR elements50; (iii) missasembly of contigs due to the presence of multiple copies of the same ISs51.ISblaNDM, we characterized the most common (pseudo-)composite transposons theoretically capable of mobilizing blaNDM , IS3000 , ISAba125 , and IS15 (28)  that could indicate the presence of cointegrate formation48 and showcase increased activity of this particular insertion element. Only 431 of the 640 putative composite transposons identified contained both complete flanking ISs, while others had at least one IS partially truncated. In addition, 1681 ISCR27, and 150 ISCR1 were found in similar proximity and appropriate orientation to mobilize blaNDM  Fig. . InteresNDM Fig. . However125 and Tn3000 were found to have a consistent length ranging from 7 to 10 Kb . After selecting only contigs with conserved transposon configurations we aligned each transposon region and identified the likely root  sequence by assessing temporal patterns , and blaNDM – dsbD with downstream ISCR1 (− strand) ending with an unknown putative IS (labelled IS-?). However, no significant temporal signal was recovered for these events.We estimated the relative timing of the formation of the Tn125 transposon carrying the blaNDM gene dated to before 1990  of the Tn990 Fig. . While tium Fig. . These fblaNDM-positive isolates. These already encompass seven bacterial species, collected in eight countries spanning four geographic regions . Such a wide host and geographic distribution, even in the earliest available genomes, illustrates the extraordinarily high mobility of blaNDM at this stage and is consistent with our molecular dating estimates.The earliest samples in our dataset are from 2007 to 2010 and comprise 21 blaNDM’s rapid spread after 2005 (coinciding with the first published observations), we measured diversity over time for several metadata categories, including country, genera, plasmid backbone and IS presence , NCBI’s GenBank34 (accessed on 15 April 2021), and EnteroBase (accessed on 27 April 2021)35, respectively. The EnteroBase repository was screened for blaNDM using BlastFrost (v1.0.0)61 allowing for one mismatch. In addition, we used the Bitsliced Genomic Signature Index (BIGSI) tool (v0.3)62 to identify all SRA unassembled reads that carry the blaNDM gene. At the time of writing, a publicly available BIGSI demo did not include sequencing datasets from after December 2016. Therefore, we manually indexed and screened an additional 355,375 SRA bacterial sequencing datasets starting from January 2017 to January 2019. We required the presence of 95% of blaNDM-1k-mers to identify NDM-positive samples from raw SRA reads. This led to the inclusion of a further 882 isolates. The dataset also included 104 NDM-positive genomes from 79 hospitalized patients across China and 25 livestock farms selected from two previous studies64. These were sequenced using paired-end Illumina (Illumina HiSeq 2500) and PacBio (PacBio RS II). The sequencing reads are available on the Sequence Read Archive (SRA) under accession number PRJNA761884. All reads were de novo assembled using Unicycler (v0.4.8)65 with default parameters while also specifying hybrid mode for those isolates for which we had both Illumina short-read and PacBio long-read sequencing data. Spades (v3.11.1)66 was applied, without additional polishing, for cases where Unicycler assemblies failed to resolve.We compiled an extensive dataset of 6155 bacterial genomes carrying the blaNDM gene were identified using BLAST (v2.10.1+)44. Forty-eight contigs were found to carry more than one copy of blaNDM and were not included in our analyses and 88 contigs were excluded due to having partial (<90%) blaNDM hits. Fourteen assemblies had a single blaNDM gene split into two contigs; these 28 contigs were also excluded. Several contigs were also removed due to poor assembly quality. The filtering resulted in a dataset of 7148 contigs (6155 samples) that were used in all subsequent analyses. Of these, 958 assembled genomes were found to contain blaNDM on multiple (mostly two) contigs, each harbouring a single and complete copy of blaNDM. Even though the information about sequencing platform or assembly methods of most samples from RefSeq, GenBank and Enterobase databases could not be determined, the distribution of blaNDM-positive contig lengths 70 run with minimum blastp percentage identity of 90% (-i 0.9) and disabled paralog splitting (-s). To identify plasmid replicon types71, contigs were screened against the PlasmidFinder replicon database (version 2020-02-25)42 using BLAST (v2.10.1+)44 where only BLAST hits with a minimum coverage of 80% and percentage identity of ≥95% were retained. In cases where two or more replicon hits were found at overlapping positions on a contig, the one with the higher percentage identity was retained. Identified plasmid types were used to cluster contigs into broader plasmid groups: IncX3, IncF, IncC, IncN2, IncHI1B, IncHI2 and other her Fig. .33 and curated to include plasmids from a bacterial host and with a sequence description, which implies a complete plasmid sequence (regular expression term used: ‘plasmid.*complete sequence’)72. Mash, a MinHash based genome distance estimator73, was applied with default settings to evaluate pairwise genetic distances between contig sequences and plasmid references. Contig-reference hits with less than 0.05 Mash distance and less than 20% difference in length were retained. Additional pruning was implemented such that, for each contig analyzed, only the 10% of best scoring plasmid reference hits were retained. A table of pairwise genetic distances between contigs and references was represented as a network that was then analyzed with the infomap74 community detection algorithm implemented in igraph75 R package. Contigs were annotated according to their community membership and the network was visualized using Cytoscape76 . A discontiguous Mega BLAST (v2.10.1+)45 search with default settings was then applied against all pairs of retained contigs. This method was selected over the regular Mega BLAST implementation as it is comparably fast, but more permissive towards dissimilar sequences with frequent gaps and mismatches. BLAST hits including a complete blaNDM gene represent maximal stretches of homology around the gene for every pair of contigs. The analysis continues by considering only portions of BLAST hits: (i) the start of blaNDM gene and the downstream sequence or (ii) the end of the blaNDM gene and the upstream sequence depending on the analysis at hand: the downstream or the upstream analysis, respectively. This trimming of BLAST hits establishes blaNDM as an anchor and enables comparisons to be made across all samples.A novel alignment-based approach was used to identify stretches of homology  as well as structural variations across all contigs upstream and downstream of blaNDM gene, the threshold is gradually increased by 10 bp. At each step, BLAST hits with a length lower than the value given by the ‘splitting threshold’ are excluded. Thus, as the ‘splitting threshold’ increases, a network of BLAST hits is also pruned and broken down into components—groups of interconnected nodes (contigs). It is expected that contigs within each component share a homologous region downstream (or upstream) of blaNDM at least of the length given by the threshold. It is therefore not possible for a single contig to be assigned to multiple components. Components of size <10 are labelled as ‘Other Structural Variants’. Also, contigs that are shorter than the defined ‘splitting threshold’ and share no edge with any other contig are considered as ‘cutting short’. Similar clustering algorithms have been previously described, e.g., CAST77, but were used for a different application.A table of BLAST hits can be considered as a network (graph), where each pair of contigs  are connected by the edge weighted by the length of the BLAST hit. The algorithm proceeds with a stepwise network analysis of BLAST hits. For this purpose, a ‘splitting threshold’ was introduced. Starting from zero that represents the start/end position of blaNDM), as well as the homology breakpoint. The precision of the algorithm is directly influenced by the step size, in this case 10 bp, and the alignment algorithm, in this case discontiguous Mega BLAST. We assessed the precision of the algorithm on the tree of structural variations downstream of blaNDM  back to their matching contigs. The mapping was done using BBMap67 . Within the output SAM file, only the overhanging reads with the CIGAR string matching the [0-9]*M[0-9]*S regular expression were selected. All overhangs of reads ≥50 bp were screened against ISFinder database47.To investigate the reasons behind a number of distinctively short 125 and 73 complete Tn3000 contigs with a known collection date and harbouring blaNDM were sequentially aligned (--pileup flag) using Clustal Omega (v1.2.3)78 specifying the blaNDM-1 sequence (FN396876.1) as a profile. Each alignment was manually inspected using UGENE (v38.0)79. Quality assessment  and linear regression analyses were employed to estimate the presence of temporal signal in the alignment83 and BactDating84 to infer the date of the emergence of the two transposons. The BactDating analysis was run with strictgamma model specification and coalescent prior on the tree. All BEAST2 analyses specified a strict prior on the molecular clock with a uniform distribution from 0 to infinity and the generalized time reversible (GTR) substitution model prior. Three population size models with default settings were used in BEAST2 analyses: Coalescent Constant population, Coalescent Exponential population, and Coalescent Bayesian Skyline. Multiple general coalescent population size models and loose (default) priors were used to corroborate dating results because, to the best of our knowledge, the complexity of transposon mobility is not currently described in any models available in BEAST2. Such an approach has yielded wider, but credible confidence intervals on the dating results.Bayesian-based molecular dating approaches were implemented in BEAST2 (v2.6.0)blaNDM element, but more importantly due to lack of host structure across transposon phylogenetic trees. In addition, all BEAST2 and BactDating runs were supplied with a maximum likelihood phylogenetic tree (starting tree prior) constructed from both transposon alignments using RAxML (v8.2.12)85 with specified GTRCAT substitution model and rooted using the inferred ancestral sequences. The chosen MCMC chain lengths for BactDating and BEAST2 runs were 107 and 1.5 × 109, respectively, to ensure convergence. We evaluated effective sample sizes (ESS) of the posterior distributions using effectiveSize function implemented in coda86 R package after discarding the first 20% of burn-in , and ISs flanking the blaNDM gene. To estimate entropy of the population and to provide confidence intervals around our estimates, we use bootstrapping with replacement (1000 iterations). At each iteration, entropy was estimated for a sampled set of contigs (X) classified into n unique categories according to the following formula:P(xi) of any sample belonging to any particular category xi  is approximated using the category’s frequency. Accordingly, higher entropy values indicate an abundance of equally likely categories, while lower entropy indicates a limited number of categories.We estimated Shannon entropy (‘diversity’) for several categorizations of geodist87 R package. Exact Jaccard distance (JD) was used as a measure of the genetic distance calculated using the tool Bindash (v0.2.1)88 with k-mer size equal to 21 bp. The JD is defined as the fraction of total k-mers not shared between two contigs. JD between all pairs of contigs was first calculated on a 1000 bp stretch of DNA downstream of blaNDM start codon continuing with a 500 bp increment. At each increment, the two distance matrices (genetic and geographic) were assessed using the mantel function (Spearman correlation and 99 permutations) from the vegan89 package in R. The correlation between genetic and geographic distance, was plotted as a function of distance from blaNDM gene (Supplementary Fig. Geographic distance between pairs of samples was determined using their sampling coordinates and the Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3


Oxytocin (OXT) and arginine vasopressin (AVP) support a broad range of behaviors and homeostatic functions including sex-specific and context-appropriate social behaviors. Although the alterations of these systems have been linked with social-related disorders such as autism spectrum disorder, their formation and developmental dynamics remain largely unknown. Using novel brain clearing techniques and 3D imaging, we have reconstructed the specification of oxytocinergic and vasopressinergic circuits in the developing mouse brain with unprecedented cellular resolution. A systematic quantification indicates that OXT and AVP neurons in the hypothalamus display distinctive developmental dynamics and high cellular plasticity from embryonic to early postnatal stages. Our findings reveal new insights into the specification and consolidation of neuropeptidergic systems in the developing CNS. Madrigal and Jurado use brain clearing techniques and 3D imaging to generate high-resolution reconstructions of oxytocinergic and vasopressinergic circuits in the developing mouse brain, which are known to play a crucial role in social behaviours. They provide insight into the specification and consolidation of these circuits from embryonic to early postnatal stages of development. OXT was first identified for its systemic function in parturition and lactation4. Additionally, cumulative evidence indicated a prominent role of OXT in parental and copulatory behaviors7 and as a pro-social neuromodulator9 increasing the salience of social stimuli and regulating social behaviors as well as aggression, anxiety, fear, and trust20. Conversely, AVP has been proposed to antagonize OXT-mediated functions22 like increasing social stress23 and reducing interpersonal trust in humans25. However, this view is being revisited after several studies exposed a more complex scenario for the interplay of OXT and AVP in the central nervous system (CNS)26. Although the details of how OXT and AVP interact to modulate neuronal function remain unknown, numerous studies have revealed that alterations of OXT and AVP circuits may underlie mental disorders often characterized by deficits in social interaction such as autism31, social anxiety and aggression32, and schizophrenia35.Oxytocin (OXT) and arginine vasopressin (AVP) are evolutionarily conserved neuropeptides implicated in the regulation of complex social behaviors. These neuropeptides are synthesized in the hypothalamus where are processed into a nine amino acid peptide chain only differing in two amino acids39 which provide insightful information, but are difficult to extrapolate to circuit formation in the entire brain. Furthermore, most of the previous work has focused on the rat brain46 even though an increasing number of studies employ the mouse as an experimental model, thus highlighting the need for more accurate connectivity maps for this commonly used species.Previous work suggests that the balance between OXT- and AVP-mediated signaling is likely to determine the display of appropriate social behaviors, thus understanding their simultaneous developmental dynamics is crucial for having a complete picture of their regulatory roles. Most studies characterizing the expression of OXT and AVP projections have employed histological methods and in situ hybridization in fixed sections+ clearing technique to analyze the expression of endogenous neuropeptides. This method in combination with light sheet fluorescent microscopy has allowed us to generate 3D reconstructions of the oxytocinergic and vasopressinergic systems in the entire mouse brain from early development to adulthood. This methodology achieves cell-specific resolution allowing a systematic quantification of oxytocinergic and vasopressinergic cells. Our data revealed that OXT and AVP neurons first appear at the caudal hypothalamic regions followed by rostral areas. Interestingly, different hypothalamic nuclei show marked differences between OXT and AVP expression during development. The rostral nuclei, such as the anterodorsal preoptic nucleus (ADPN) and the periventricular nucleus (PeVN), together with neighboring areas such as the bed nucleus of stria terminalis (BNST) display a fairly homogeneous expression whereas the caudal nuclei (supraoptic nucleus (SON), paraventricular nucleus (PVN), and the retrochiasmatic area (RCH)) exhibit greater heterogeneity with a large population of neurons coexpressing both OXT and AVP during early postnatal stages. Our analysis indicates that the accessory nucleus (AN) is the most heterogeneous of the hypothalamic nuclei with a rostral section almost exclusively constituted by OXT and OXT + AVP neurons and a caudal region enriched with AVP neurons.To overcome these current challenges, we have now implemented the iDISCOAlthough the expression of OXT + AVP neurons is quite high at postnatal day seven (PN7), most of the nuclei exhibit an increase in the number of OXT neurons in detriment of the mixed OXT + AVP population during postnatal development. This switch in neuropeptide expression is particularly significant in BNST and ADPN, where the presence of AVP neurons decreases drastically in the adult brain. These developmental adaptations are expected to have functional consequences impacting the ratio of AVP/OXT innervation to their projection sites.47. These studies have confirmed our observation of a significant increase of oxytocinergic neurons in most hypothalamic nuclei as development progresses. Strikingly, some OXT positive neurons in the SON and RCH of the commercial OXT-Cre line are not recognized by standard OXT antibodies suggesting the presence of nucleus-specific OXT precursors.In addition to wild type animals, we have characterized OXT expression in a commercial OXT-Cre mouse lineIn summary, our in-depth circuit analysis has revealed that OXT and AVP expression exhibits distinct developmental dynamics in the mouse brain. These dynamic adaptations are likely to modulate the functional properties of different brain regions according to their developmental stage, thus contributing to the refinement of the neuronal circuits that support context-appropriate social behaviors later in life.35. Most of OXT and AVP innervation in the CNS comes from the hypothalamus, a brain area comprising highly heterogeneous nuclei. Pioneer work using in situ hybridization and immunohistochemistry techniques described with great detail the hypothalamic circuit in the developing rat brain46, however, the mouse brain attracted less attention partly because of its smaller size which hinders the identification of the intricate hypothalamic nuclei. Because an increasing number of studies particularly on social behavior utilize mouse models, it is necessary to improve our understanding of the development of OXT and AVP circuits in this species.Understanding the formation and developmental dynamics of oxytocinergic and vasopressinergic circuits is of great significance given their regulatory role in social behaviors. The identification of alterations during the development of these systems is expected to reveal critical insights into the underlying causes of several neuropsychiatric disorders50. Here, we implemented a protocol for 3D imaging of solvent-cleared brains, adapted from the iDISCO+ protocol described in Renier et al.51. Incubation time for anti-OXT and anti-AVP antibodies was optimized according to brain size and tissue firmness. Best results were obtained with incubation times of 5 days for E16.5 brains, 10 days for PN0, 2 weeks for PN7, and 3 weeks for adult brains. In our hands, these modifications resulted in fully cleared brains . This method in combination with 3D image processing, enabled isolation of single hypothalamic nuclei . This reduction is also observed in AVP-dominant nuclei like the SCH53 where the percentage of AVP-expressing cells decreases from early postnatal stages (PN0) to adulthood . This phenomenon is quite prominent in some nuclei, such as ADPN and neighboring areas like the BNST, where the percentage of neurons co-expressing OXT and AVP decreases in the adult brain, although in a rather heterogeneous fashion . In the rest of the nuclei, co-labeling of OXT and AVP reaches a peak at PN7 that thereafter steadily decreases over time .Quantification of 3D imaging revealed that at early postnatal stages most hypothalamic nuclei exhibit a high percentage of neurons co-expressing OXT and AVP  used in iDISCO+ experiments . Thus, the lack of colocalization between OXT and tdTomato in this mouse line, may suggest changes in the internal program of OXT positive cells likely due to functional changes during embryonic and early postnatal development58. This heterogeneity is developmentally regulated as co-labeling increases as maturation progresses . However, these early OXT precursors persist until adulthood in particular areas like the SON and the RCH  express a Cre recombinase sequence right after the stop codon of the OXT gene and they are an effective tool for identifying OXT neuronsibody PS3 used in nts Fig.  with no ibody PS3 used in ses Fig.  to reachses Fig. ii as preRCH Fig.  and a hiRCH Fig. . These f20. Our work aims to overcome these current challenges by providing a detailed analysis of the OXT and AVP systems in the developing mouse brain. Here, we have implemented novel iDISCO+ clearing techniques to generate high cell-resolution brain-wide maps of the developing OXT and AVP systems. Systematic 3D cell quantification revealed that the formation of these circuits exhibits specific features distinct from the rat and other rodents which need to be considered when working with this animal model.Until now, a complete picture of the OXT and AVP innervation in the developing mouse brain has been lacking whereas more detailed information has been available for the rat brain. This lack of knowledge has prevented establishing meaningful correlations between anatomical and functional data obtained from transgenic mouse lines and mouse behavioral studies59, mouse OXT and AVP are mostly synthesized in the PVN, SON, and AN. However, vasopressinergic and oxytocinergic cells can be found in other hypothalamic areas as well as non-hypothalamic regions, where these systems have been less studied. The cellular resolution achieved by the iDISCO+ clearing method revealed OXT and AVP neurons in non-hypothalamic areas like the MeA and particularly, the BNST  that specifically recognizes OXT-neurophysin I39. Immunohistochemistry and in situ hybridization studies in the rat brain indicated the existence of several intermediate OXT forms during early development59. In this regard, the analysis of a transgenic OXT mouse line (OXT-tdTomato) revealed a significant percentage of oxytocinergic neurons that are not recognized by anti-OXT PS38  until adulthood  which exhibit a Cre reporter allele with a loxP-flanked STOP cassette preventing transcription of the red fluorescent protein variant tdTomato inserted into the Gt(ROSA)26Sor locus. OXT-tdTomato mice express robust tdTomato fluorescence following Cre-mediated recombination. All experiments were performed according to Spanish and European Union regulations regarding animal research (2010/63/EU), and the experimental procedures were approved by the Bioethical Committee at the Instituto de Neurociencias and the Consejo Superior de Investigaciones Científicas (CSIC). Animals were housed in ventilated cages in a standard pathogen-free facility, with free access to food and water on a 12 h light/dark cycle.Experiments were performed in wild type ICR mice bred in-house. Brains were extracted at E16.5, PN0, and PN7 from young female adults (2–3 months). Data from female and male animals were pooled for E16.5, PN0, and PN7 animals. A limitation of the study is the use of a mixture of male and female brains to analyze early developmental stages  hindering the detection of potential sex differences. Oxytocin-Ires-Cre:Rosa26iDTR/+OXT-Cre (OXT-Cre thereafter) mice were obtained from Jackson Laboratories (strain ID 024234). These animals express Cre recombinase under the control of the endogenous OXT promoter and has been used previously to specifically target oxytocinergic neurons59 ; rabbit anti-oxytocin ; rabbit anti-RFP ; rat anti-RFP ; rabbit anti-vasopressin . Note that mouse anti-OXT (PS38) and rabbit anti-OXT label a highly overlapping pool of OXT neurons in the adult brain  in PBS overnight. Embryos were fixed overnight by immersion in 4% PFA. Samples were embedded in agarose (4%) and sectioned at 50 µm using a Leica VT1000 S vibratome. Sections were rinsed three times in PBS and incubated 1 h in PBST . Samples were incubated overnight at 4 °C in a rotating shaker with the following primary antibodies: mouse anti-oxytoxin PS38+ optical clearing was performed as described in Renier et al.,50 and Renier et al.,51. Mice were intracardially perfused and brains were fixed in 4% PFA in PBS overnight. Embryos were fixed in 4% PFA immersion overnight. Brains were dehydrated using a series of concentrations of methanol  and then incubated in 6% H2O2 in methanol overnight in order to bleach the samples. After rehydratation, samples were blocked with PBS-GT  for durations that depended on developmental stage: overnight for E16.5 brains; 2 days for PN0 and PN7 brains; and 4 days for adult brains. Brains were incubated with OXT and AVP primary antibodies  and rabbit anti-vasopressin, Millipore PC234L (1:2000)) diluted in 0.1% saponin  PBS-GT solution for 5 days (E16.5), 10 days (PN0), 2 weeks (PN7) or 3 weeks (adult brain) at 37 °C on a rotating shaker. Afterward, samples were incubated with secondary antibodies directly conjugated to a fluorophore donkey anti-mouse Alexa-647  or goat anti-rabbit Alexa-594  at 37 °C overnight or two days for adult brains. Immunostaining controls using secondary antibodies (without primary antibodies) were performed to test the specificity of the primary antibodies for OXT and AVP.Whole-mount immunostaining and iDISCO2O at RT in a rotating shaker. Shaker incubations were for 1 h for embryonic and postnatal brains, and 1.5 h for adults. Specimens were then incubated twice in 100% methanol for 1–1.5 h, and treated overnight in 1 volume of 100% methanol and l/3 volumes of 100% dichloromethane . On the next day, brains were incubated in 100% DCM for 30 min. Lastly, samples were cleared in 100% dibenzylether  until becoming translucent.For the clearing, brains were dehydrated in 20, 40, 60, and 80% methanol in HConfocal microscopy was performed with a laser scanning Leica SPE-II DM5550 microscope with ×10, ×20, and ×40 objectives. Brains were imaged on a bidirectional triple light-sheet microscope  equipped with a binocular stereomicroscope  with a ×2 objective  using a 5.7 mm working distance dipping cap. Samples were placed in an imaging reservoir made of 100 % quartz (LaVision BioTec) filled with ethyl cinnamate . The step size in Z-orientation between each image was fixed at 3 μm for 2.5× and 4× magnifications for nuclei identification and cell counting, and 5 μm for 0.63×, 1×, and 1.6× magnifications for whole-brain images analysis. 3D imaging was performed using ImspectorPro software (LaVision BioTec) and processed by the Imaris x64 software . Video analysis was optimized for cell identification.89. The volume estimation of each nucleus was generated using immunohistochemical labeling as a guide and the Surface Imaris tool. False colors were assigned to these brain areas with the Surface Imaris tool. High-quality 3D images were selected for cell quantification. For accurate quantification of AVP- and OXT-expressing cells the fluorescence threshold was set manually and false positives were discarded from the final analysis using Imaris Spots tool. It is notable that at embryonic stages the image resolution, is not as sharp due to a greater abundance of blood vessels and neuronal processes. 3D pictures and movies were generated with Imaris. Cell 3D positioning was achieved by the statistical data-visualization module of Imaris, vantage view. High dimensional color plots were generated to analyze the 3D distribution of each cell population in the three axes .Quantifications of OXT and AVP cells in each hypothalamic nucleus were calculated using alternate hemispheres of four brains. Brains at different developmental stages were processed and quantified using Imaris x64 software  using the tools for 3D reconstruction and segmentation. Boundaries of hypothalamic nuclei were identified according to the mouse brain atlas of Franklin and Paxinost-test. P < 0.05 was considered statistically significant.Several rounds of experiments were performed to assure the accuracy of the results. Samples were processed simultaneously and measurements were performed once the staining protocols were completed in no particular order. The authors were not blind to the age of animals during the processing of the samples since the size of the brain correlates with the age. However, the authors were blind during the analysis. Data were analyzed with Imaris and exported to Excel  for data handling. Sample size were estimated using the power analysis set to significance level <0.05 and 0.80 to achieve over 80% of power with n = 4 samples per group. For statistical analysis, cell counts were estimated to follow a negative binomial distribution and adjusted to account for false positives. Samples were discarded when exhibiting poor immunostaining or deficient clearing. Results in the bar graph plot are expressed as the mean ± S.E.M. Statistically significant differences were determined using an unpaired Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4Supplementary Video 5Supplementary Video 6Reporting Summary


BackgroundBrachioradial pruritus (BRP) is a neuropathic dysesthesia manifesting as pruritus over the dorsolateral forearm. While the etiology is unknown, intensive sun exposure and cervical spine disease have been proposed. This study describes the clinical, electrodiagnostic (EDX), and cervical MRI findings in nine patients diagnosed with BRP.Materials and methodsAll patients underwent EDX and cervical MRIs. Numerous metrics were documented including presenting symptoms, neurological examination, EDX findings, and cervical MRI features.ResultsAll nine patients experienced pruritus of the arms/forearms, typical of BRP, which was unilateral in eight (89%) cases. Decreased pinprick sensation was noted in the arms/forearms (five [56%] patients) or of the thumbs, index, and/or middle fingers (four [44%] patients). Four (44%) patients had either decreased or absent biceps and brachioradialis deep tendon reflexes (DTRs), while one (11%) patient had decreased triceps and brachioradialis DTRs. The EDX revealed abnormalities in eight (89%) patients. Increased polyphasic units, decreased motor units, and/or denervation changes were recorded by needle electromyography (EMG) in eight (89%) patients: the biceps in seven (88%) and both the brachioradialis and triceps in four (50%) patients. The EMG abnormalities indicated chronic radiculopathy involving C6 in six patients and C5 and C6 in one patient. All nine patients had cervical spine disease, encompassing disc protrusions, spondylosis, spinal stenosis, and/or foraminal stenosis.ConclusionsBRP in this series of patients was accompanied by chronic cervical radiculopathy involving predominantly C6 and C5. EDX and cervical spine MR imaging should be considered essential investigations in the evaluation of patients with BRP. Brachioradial pruritus (BRP) is a neuropathic pruritus marked by itching, burning, stinging, and tingling over the dorsolateral aspect of the arms and forearms -8. The pThe diagnosis of BRP may prove challenging as patients often undergo invasive skin biopsies and are evaluated by various specialists, most frequently by dermatologists as well as neurologists and pain medicine physicians . The “icWe report nine patients who were diagnosed with BRP, all of whom underwent EDX and a cervical MRI. The presenting symptoms, physical examination, cervical MRI, and EDX findings in these nine patients are featured. The possible association between BRP and cervical spine pathology and treatment is discussed. The pathophysiology of BRP and the importance of EDX in the diagnostic evaluation of BRP are also discussed.Under an Institutional Review Board (IRB)-approved protocol, we performed a 10-year (2012-2021) retrospective analysis of patients referred to our Neurodiagnostic Center for EDX studies to evaluate the presence of upper limb neuropathy such as carpal tunnel syndrome or cervical radiculopathy. Over this 10-year period, approximately 15,000-20,000 patients were referred to our facility for EDX studies. The EDX physician diagnosed the nine patients with BRP based on the pre-test clinical evaluation. Except in one patient, the referring physician did not consider a diagnosis of BRP. The patients underwent our protocol of an initial neurological examination followed by nerve conduction and electromyography (EMG) studies. BRP was not diagnosed based on the EDX studies. The EDX studies and cervical MRIs were used to determine whether the patients with BRP had underlying cervical radiculopathy (C5-C6 nerve roots which supply the area of pruritus).The clinical history evaluated the onset and progression of symptoms, with identification of paroxysms of severe pruritus involving the dorsolateral forearm and or upper arm. Details regarding the nature and topography of sensory symptoms including pruritus were obtained, including triggering/worsening and relieving factors. The presence and distribution of cervical radicular symptoms were documented. The neurological examination was aimed at mapping out the area of sensory disturbance  along with the evaluation of muscle strength, tone, and reflexes. The presence of skin lesions secondary to pruritus was also assessed.The EDX studies were performed in our American Association of Neuromuscular & Electrodiagnostic Medicine (AANEM)-accredited facility using standard protocol of our laboratory . Motor cInformed consent was obtained from all patients. The University of Louisville IRB determined that our study was exempt according to 45 CFR 46.101(b) under Category 4. The IRB number is 21.0796.Clinical findings and neurological examination of patients with brachioradial pruritusNine patients were diagnosed with BRP based on presenting symptoms , neurological examination, and EDX findings Table .BRP was suspected in only one of these patients prior to the referral for EDX. The mean age was 57.5 years (range: 47-71 years), and seven (78%) patients were female. All nine patients complained of severe pruritus of the upper extremities which was unilateral in eight (89%) cases. None of the patients had radicular pain on presentation; two patients had a radicular tingling sensation along with the intense pruritus. The patients did not report any predisposing or inciting factors such as prolonged sun exposure prior to the BRP diagnosis, however, they often stated that the symptoms were worse in the summer than in winter.Decreased pinprick sensation was noted in the arms/forearms (five [56%] patients) or in the thumbs, index, and/or middle fingers (four [44%] patients) Table . Four , decreased motor units, and/or denervation changes were recorded by needle EMG in eight (89%) patients: the biceps in seven (88%) patients and both the brachioradialis and triceps in four (50%) patients. The EMG abnormalities were consistent with chronic radiculopathy involving C6 in six patients and at C5 and C6 in one patient.Cervical MRI findings in patients with brachioradial pruritusAll nine patients had evidence of cervical spine disease, encompassing disc protrusions, spondylosis, spinal stenosis, and/or foraminal stenosis Table . Only onFollow-up of patients with brachioradial pruritusThe nine patients with BRP attained variable improvement of their symptoms with medications , with gabapentin providing the most relief. One patient underwent cervical epidural injections and acupuncture for BRP without benefit.While it is unclear whether cervical radiculopathy is the major causative factor in BRP , 18, 19,The cervical origin of BRP has also been supported by myriad treatments aimed at the cervical region with ensuing relief of pruritic symptoms, including cervical spine manipulation, physiotherapy, and cervical nerve root blocks , 14, 19.It has been proposed that BRP is caused by either peripheral nerve damage (induced by solar radiation) or from sensory pathways involving the spinal cord and/or cervical nerve roots  . A-deltaThe mechanism by which BoNT acts on neuropathic pain involves inhibiting the secretion of pain mediators  from nerve endings and dorsal root ganglia (DRG), decreasing local inflammation around the nerve endings, deactivating the sodium channel, and demonstrating axonal transport . BoNT reEDX has not been routinely done in the investigation of patients with BRP . As nervOur case series illustrates the importance of adding electrodiagnostic data to the clinical and MR imaging findings to arrive at the cause of BRP. The presence of MR and EDX findings supportive of cervical radiculopathy in all of our patients further substantiates the role of cervical spine disease in BRP. While the underlying pathophysiology is unclear, the discovery of selective neural pathways for itch and pain suggests the possibility that spinal root/cord lesions may lead to the predominance of pain or itch sensation. While BRP is a clinical diagnosis and may respond to symptomatic treatment of pruritus, the presence of additional neurological findings suggestive of a cervical root or cord lesion should prompt further workup including imaging studies of the cervical spine and, if indicated, EDX studies.


The sample consisted of 3892 adolescents . Parents’ extremely negative reaction towards ENDS when they found their children using ENDS (AOR = 0.309) was connected to a lower risk of oral health issues. The findings suggest that clinicians and policymakers need to consider the roles of these factors when developing strategies to improve oral health outcomes.The influence of familial and social environments plays a significant role in Electronic Nicotine Delivery System (ENDS) use and may contribute to poor oral health among adolescents. This study utilized the Population Assessment of Tobacco and Health (PATH) database and included youths aged 12 to 17 years who reported no history of dental health issues at baseline. Adjusted odds ratios (AOR) were used to examine the association between END-related familial factors and oral health among adolescents in the United States, with statistical significance set at  The past few decades have seen a marked decrease in cigarette use in the United States . HoweverAlthough marketed as less harmful than combustible tobacco products, research indicates that ENDS use adversely affects health. ENDS solutions contain toxic compounds such as lead and nickel  and mostEvidence implicates familial factors as strong predictors of smoking uptake among adolescents. Having parents , siblingAlthough research links familial smoking behaviors and attitudes with smoking  and ENDSThe Population Assessment of Tobacco and Health (PATH) study is a nationally representative longitudinal study of 45,971 United States adults (18 years and older) and youth (12–17 years). PATH collects self-reported information on tobacco-use patterns and associated health behaviors through audio computer-assisted self-interviews to examine tobacco use. PATH is an ongoing longitudinal study, and its data collection began with Wave 1 in 2013, with five subsequent data collections for youth participants from Wave 1 to Wave 5. All wave 1 respondents stayed eligible for follow-up interviews if they remained residents of the US and were not incarcerated.http://doi.org/10.3886/Series606 (Accessed on 16 January 2022).PATH uses sampling weights to develop population estimates representative of the noninstitutionalized United States population. Youths who participated in three waves of the PATH study (19 October 2015–23 October 2016 (Wave 3), 1 December 2016–3 January 2018 (Wave 4), 1 December 2018–30 November 2019 (Wave 5)) were eligible for the present study. Details regarding PATH survey interview procedures can be found at Since the variables from waves 1 and 2 were different than those from waves 3, 4 and 5, this study excluded waves 1 and 2’s data. The inclusion criteria for sample selection were: (1) Youths who were 12 to 17 years old in all three waves ; (2) Youths who were 17 years old at wave 4 and aged into adults (18 years old) at wave 5; (3) Youths who were 17 years old at wave 3 and aged into adults (18 years old) at wave 4; and (4) Shadow youths who were 10 to 11 years old at wave 3 and aged into youths (12 years old) at wave 4. This yielded an eligible sample of 15,449 individuals prior to applying exclusion criteria. The exclusion criteria were: (1) Individuals with dental problems at baseline; (2) Individuals with missing data for the outcome variable; and (3) Individuals with missing data for the weight variable. After applying the exclusion criteria, the final sample size for this study was 3892. Socio-demographics. Socio-demographic variables consisted of age , gender , ethnicity (Hispanic and non-Hispanic), race , and annual household income. Income was collapsed into the following categories: Less than USD 10000, USD 10000–USD 24999, USD 25000–USD 49999, USD 50000–USD 99999, and USD 100000 or more. Outcome Measure. The outcome measure of interest was the self-reported response from Wave 5 concerning dental health issues diagnosed in the past year. Specifically, it asked the question: In the past 12 months, have you ever been told by a doctor, dentist, or another health professional that you have dental health issues? (Yes/No).Familial variables. Five parental dichotomous variables related to ENDS use were assessed at baseline (Wave 3): (1) In the past 12 months, have your parents or guardians talked with you, even once, about not using ENDS? (Yes/No); (2) Do you think any tobacco products or ENDS might be available to youths at their parent or guardian’s home? (Yes/No); (3) Rules about using ENDS inside the home ; (4) Have close biological relatives ever been troubled by SUD? (Yes/No); and (5) If your parents or guardians found you using ENDS, how do you think they would react? (Be very upset/Have no reaction or not be so upset). Risk factors. Alcohol use, drug use, and tobacco use, all of which are considered risk factors for poor oral health [l health . The alcp < 0.05.Descriptive statistics of participants’ baseline socio-demographics, familial factors, and risk factors were calculated using Binary logistic regression models to assess associations between ENDS-related familial factors at baseline (Wave 3) and oral health outcomes at follow up (Wave 5)—controlling for socio-demographics, alcohol use, and tobacco use. Sensitivity analysis was conducted by examining recent use (within the past 12 months) and lifetime use (ever use) of alcohol and tobacco. Variables that had less than 5% of cases in any single response category were not included in regression analyses due to a sparse data matrix. To obtain nationally representative estimates, the Wave 5 ‘all-wave’ weights were applied to the sample estimates. Adjusted odds ratios (AOR) and 95% confidence intervals (95% CI) were reported. All analyses were conducted with IBM SPSS Statistics V28 software, and statistical significance was set at Adolescents with a household income of less than USD 10000 had a higher likelihood of developing dental health issues than those households earning greater than USD 100000 per year . After cThis study used a nationally representative PATH database to examine the impact of ENDS-related familial factors on the oral health of adolescents in the United States. It represents the first study examining what familial factors, if any, related to ENDS use impact an adolescent’s oral health. The results indicate that the lack of a negative parental reaction if found using ENDS emerged as an independent predictor for poorer oral health. The findings reported here suggest that addressing familial factors offers an opportunity to improve the oral health of adolescents. Good oral health depends on the dynamic interactions between an individual and their cultural, psychological, social, economic, and political environment. For children and teens, health depends on a strong family environment. Our findings indicate that opportunities may exist to strengthen the familial environment in ways that support oral health at both the provider level and in the public health domain.Cigarette smoking significantly increases the risk of periodontal disease ,39,40. SOur study found that those with a household income of less than USD 10,000 were more likely than those households earning greater than USD 100,000 per year to report dental problems, but this may be due to higher income earners’ ability to pay for comprehensive dental care, whereas those with low incomes may delay visiting a dentist until they experience dental problems . FurtherThe results also suggest that public health measures addressing familial factors offer an opportunity to improve oral health. Public health dentistry focuses on the community rather than the individual to prevent and control dental disease . ProgresThere are several limitations to this study. First, the data were self-reported, and participants might provide socially desirable responses rather than their true responses. Another limitation is that the last data collection occurred in 2019, and recent legislative changes and publicity about adverse ENDS effects might yield different results if more recent data were available. A third limitation is that the PATH survey contains limited response options, but more detailed response options could provide better insight.In contrast, a study strength is that it used a database representative of the United States youth population. In addition, assessing longitudinal data allowed the selection of youths without dental problems at baseline, to then identify if over time the presence of ENDS-related familial factors predicted future oral health.This study used the PATH database to examine the longitudinal effect of ENDS-related parental factors on oral health and found that the lack of a negative parental reaction if found using ENDS adversely affected oral health in adolescents and emerged as an independent risk factor for poor oral health. These findings suggest that strengthening the family environment at both the provider level and in the public health domain may improve the oral health of adolescents.


Cerebral Cortex, the group of Heiko Luhmann offered exciting new insights into the neurophysiological underpinnings of neurodevelopmental disorders, which exhibit seizures and autistic behavior as clinical hallmarks [TSC1 or TSC2, which encode the mTOR complex 1 (mTORC1)-inhibiting proteins TSC1 (hamartin) and TSC2 (tuberin), respectively [In a recent paper published 2020 in allmarks . In thatallmarks . Importaallmarks , 8. On tectively . How abeTSC1 in principal forebrain neurons [TCS2 than TSC1 [Previous work from the group of Bernardo Sabatini in another mouse model of TSC showed that synaptic inhibition of hippocampal pyramidal cells is diminished through a postsynaptic mechanism, whereas the strength of glutamatergic neurotransmission remained unaffected, pointing to an imbalance between excitation (E) and inhibition (I) . It is w neurons . By conthan TSC1 .TSC2 (Tsc2+/− mice) and, with their interest in autism spectrum disorders, focused their electrophysiological interrogation on acute brain slices of the medial prefrontal cortex (mPFC) [B receptors [Consequently, the Luhmann lab explored a mouse model with haploinsufficiency of x (mPFC) . Most imeceptors . An intrPflügers Archiv—European Journal of Physiology, Bassetti, who was also lead author on the preceding paper, and his colleagues elaborate on their earlier findings with a particular emphasis on how pharmacological activation (with baclofen) or inhibition (with CGP55485) of presynaptic GABAB receptors affects the frequency of miniature and spontaneous EPSCs and IPSCs in layer 2/3 pyramidal cells of the mPFC of Tsc2+/− and wt mice 25–40 days old [+/−, but not in wt slices, suggesting that GABAB autoreceptors on GABAergic terminals are not tonically activated by ambient GABA in the mutant preparation.In the paper published in this issue of B receptor activation as a treatment option for neural hyperexcitability in neurodevelopmental disorders. In summary, Bassetti and colleagues have introduced a novel and unexpected pathophysiological mechanism that substantially furthers our understanding of the complex cellular and circuit alterations in a mouse model of TSC.In further experiments on evoked IPSCs and spontaneous EPSCs and IPSCs, the authors found that GABAergic terminals in mutant slices responded more sensitive to baclofen than those in control slices, thereby shifting the E/I ratio towards excitation in the former, whereas this parameter was hardly affected in the latter. The resulting net excitatory effect of baclofen on prefrontal neurons sends a note of caution when considering GABA