This feature is suggested to allow for the CB to function as an acute O2 sensor, being stimulated and activating systemic protective reflexes before the metabolism of other cells becomes compromised. What is less clear is precisely how a fall in mitochondrial activity links to type I cell depolarisation, a process that is required for initiation of the chemotransduction cascade and post-synaptic action potential generation. Multiple mitochondrial/metabolic signalling mechanisms have been proposed including local generation of mitochondrial reactive oxygen species (mitoROS), a change in mitochondrial/cellular redox status, a fall in MgATP and an increase in lactate. Although each mechanism is based on compelling experimental evidence, they are all not without question. The current review aims to explore the importance of each of these signalling pathways in mediating the overall CB response to hypoxia. We suggest that there is unlikely to be a single mechanism, but instead multiple mitochondrial related signalling pathways are recruited at different PaO2s during hypoxia. Furthermore, it still remains to be determined if mitochondrial signalling acts independently or in partnership with extra-mitochondrial O2-sensors.It is generally acknowledged that the carotid body (CB) type I cell mitochondria are unique, being inhibited by relatively small falls in P This alll types . Of the ll types  and it h2 binding within mitochondria takes place within the CuB/haem a3 (cytochrome a3) binuclear centre of cytochrome c oxidase (complex IV). Multiple groups have shown that the CB mitochondrial electron transport chain is inhibited as the O2 drops below an unusually and uniquely high PO2 threshold  -positive CB type I cells diminishes both the type I cell and ventilatory response to hypoxia in the mouse  and flavoprotein (Fp) redox status  and Ndufs2 deficient type I cells. Consistent with the idea that complex I derived ROS is an important effector signalling molecule, mice with conditional or inducible deletion of Ndufs2 (Ndufs2-KO) displayed a significantly attenuated rise in respiratory frequency when challenged by acute exposure to 10% environmental O2 , did not impair CB or ventilatory responses to hypoxia  can be achieved using ascorbate and TMPD following blockade of upstream mitochondrial complexes  gained significant attention due to its association with mediating cardiac myocyte cell death following ischemia-reperfusion . The str I cells . Howeversitivity .2+]i during hypoxia. A similar depletion of hypoxic sensitivity was observed in PASMCs isolated from mice expressing a tunicate alternative oxidase, which allowed for electron flux to continue during concomitant inhibition of mitochondrial complexes III and IV  did lead to chemoafferent stimulation in the isolated rat CB, but this measured only approximately 10–30% of that induced by a subsequent severe hypoxic stimulus . AdditioI and IV . It will2 range approximately equal to that which is known to stimulate the type I cell H . The rise I cell  and is de I cell . An elev systems . In the currents . The ide2 and FMNH2 are key sources of electrons for both mitochondrial and cytosolic redox reactions. Thus, their relative concentration in comparison with their oxidised forms  can be used to indicate the overall mitochondrial and/or cellular redox status  which in turn promotes NADPH oxidase activity. Accordingly, pharmacological inhibition of PIN1 was found to partially inhibit membrane depolarisation caused by hypoxia. However, mice lacking the Gp91 phox gene exhibit full CB and ventilatory sensitivity to hypoxia (NAD(P)H, FADHx status . In the x status . As Fp f hypoxia . Thus, t2+, forming MgATP . It does therefore appear that MgATP falls in the rat type I cell upon exposure to hypoxia. However, it is not apparent if this takes place over the same physiological PO2 range known to stimulate the type I cell, and further studies are needed to validate results in other species including mouse and human.To date, no study has been able to make direct measurements of type I cell ATP or MgATP. However, using Mag-indo-1, a Mg hypoxia . They co+ channels, rises in [Ca2+]i and chemoafferent excitation , whose phosphorylation (and thus activation) status is enhanced by an increase in the AMP/ATP ratio. Robust expression of AMPK subunits have been reported in both rodent type I cells  and humalication . Compounon  (When oxidative phosphorylation becomes restricted during low Ohibitor) . To mainhibitor) . Exaggerhibitor) .+ currents . Another group has reported contradictory results, whereby single CB type I cells isolated from Olfr78−/− mice produced neurosecretory responses to hypoxia that were indistinguishable from WT littermates  are expressed in type I cells, but not MCT-1, providing potential route for lactate both into and out of the cells . In isolecretion . This co2 sensing but do suggest that it most likely does not act through OLFR78. If lactate does reach high mM concentrations in CB tissue during hypoxia, then it could act by alternative mechanisms such as causing intracellular acidification  have an almost completely absent hypoxic ventilatory response (2S in hypoxia is thought to be a consequence of reduced CO synthesis subsequent to a decrease in the haem-oxygenase 2 activity (2S is able to rapidly and reversibly inhibit BKCa channels (2S required to excite isolated rat type I cells also evoke an increase in NADH, consistent with concurrent mitochondrial inhibition (2S in modifying the O2 sensitivity of the unique CB type I cell mitochondria requires further exploration as does the intriguing possibility of a novel functional role of mitochondrial generated reactive sulphur species (Mitochondrial signalling may act either independently or in partnership with extra-mitochondrial On of H2S . Compellresponse . CBs isoactivity . Althougchannels  it is alhibition . This co species .2 leads to disruption of transporter activity involved in maintenance of intracellular pH (2-O2 stimulus interaction. Clearly, experimental data is now required to validate these model data. Given the emerging prominence of the CB hyperactivity in initiating hypertension and cardiac arrhythmia (2 sensitivity and signalling is altered in pathology.In this review we have discussed some of the key mitochondrial related signalling mechanisms in the CB known to date. It is likely that novel mitochondrial and extra-mitochondrial signalling mechanisms will emerge in the future as we move towards a more complete understanding. Indeed, very recent data suggests that local mitochondrial temperature transients contribute to type I cell membrane depolarisation . Furtherlular pH . The ensrhythmia  future s


Saccharibacteria strains retrieved from the oral microbiome. MAGs were obtained from nonstimulated saliva samples from hosts with various clinical statuses and correspond to distinct species taxonomically placed within the Saccharimonadaceae family, as determined by genome-wide analysis against previously described TM7 genomes.We report the draft metagenome-assembled genomes (MAGs) of five putatively novel  Saccharibacteria species harbor very peculiar genomes that might have coevolved as a reflection of their epibiontic lifestyle with other oral bacteria  are ultrasmall organisms that were originally detected in the open environment , some ofbacteria . In thisVolunteers were recruited at the Dental Clinic of the Amazonas State University (Brazil) with no distinctions with respect to gender, race, or age. Written informed consent forms were signed by participants in accordance with the 7th version of the Declaration of Helsinki (2013). One-milliliter nonstimulated saliva samples were collected, subjected to DNA extraction with the MasterPure complete DNA and RNA purification kit (Epicentre) following the manufacturer's instructions, and quantified via a Qubit fluorometer (Thermo Fisher Scientific). Metagenomic DNA was mechanically fragmented with a proprietary hydrodynamic sonication protocol (Novogene HK), and 1.0 μg DNA was used for library preparation with the NEBNext Ultra DNA library preparation kit (New England Biolabs). Products in the range of 300 bp were selected and sequenced in a HiSeq 2500 instrument (Illumina) with a HiSeq SBS v.4 kit targeting 150-bp paired-end reads. Raw sequence quality control was performed with readfq_meta v.8 software (Novogene HK) by removing reads displaying more than 40 bp with Q values of ≤38 or presenting more than 10 undetermined (N) base pairs. Paired-end reads were merged and adapter sequences were removed with PEAR v.0.9.8. Human DNA was filtered out via genome mapping with Bowtie2  accordinSaccharibacteria species. General DNA sequencing statistics and annotation features of the announced MAGs are presented in Of the 27 samples analyzed, only 5 yielded successful genome assemblies according to the adopted parameters, with clones OHS0006, OHS0013, and OHS0010 corresponding to healthy subjects and clones OPS0014 and OPS0017 to generalized chronic periodontitis cases. The reported genomes displayed <95% ANI values among themselves and with the 31 TM7 reference genomes available at GTDB, suggesting a direct correspondence to potentially novel The final drafts of MAGs reported in this study, along with the respective raw sequence reads and BioProject and BioSample information, are publicly available at DDBJ/EMBL/GenBank under the accession numbers presented in


Following publication of the original article , the refThe in text citation for the text has been changed fromAdditionally, when the extensiveness of mediastinal lymph node dissection is increased, i.e. with transcervical extended mediastinal lymphadenectomy (TEMLA) or video-assisted mediastinoscopic lymphadenectomy (VAMLA) techniques, the risk of RLN injury also rises [4]. In a contemporary series with 108 patients after VAMLA, recurrent nerve palsy was identified in 5% of patients [5].toAdditionally, when the extensiveness of mediastinal lymph node dissection is increased, i.e. with transcervical extended mediastinal lymphadenectomy (TEMLA) or video-assisted mediastinoscopic lymphadenectomy (VAMLA) techniques, the risk of RLN injury also rises [5]. In a contemporary series with 108 patients after VAMLA, recurrent nerve palsy was identified in 5% of patients [6].The in text citation for the text has been changed fromChart review  was performed retrieving data on preoperative characteristics including demographics and oncological details (if appropriate), intraoperative records regarding harvested lymph node (LN) stations during CM, and postoperative data including voice changes and clinical follow-up. Voice changes with full recovery within 14 days were attributed to intubation-related causes [6].toChart review  was performed retrieving data on preoperative characteristics including demographics and oncological details (if appropriate), intraoperative records regarding harvested lymph node (LN) stations during CM, and postoperative data including voice changes and clinical follow-up. Voice changes with full recovery within 14 days were attributed to intubation-related causes [7].The in text citation for the text has been changed fromIn addition, the standardized 30-item self-administered Voice Handicap Index (VHI) questionnaire was used to assess the impact of voice impairment on a patient’s QoL . A 4-point interval score from ‘never’ (0 points) to ‘always’ (4 points) is used to indicate the frequency of various voice complaints. These items can be combined into a total score, and they can be counted separately into three different domain subscores , with higher scores indicating more severe voice impairment. In addition, cut-off points have been proposed  to categorize the various scores into either mild, moderate or severe voice impairment [7].to.In addition, the standardized 30-item self-administered Voice Handicap Index (VHI) questionnaire was used to assess the impact of voice impairment on a patient’s QoL . A 4-point interval score from ‘never’ (0 points) to ‘always’ (4 points) is used to indicate the frequency of various voice complaints. These items can be combined into a total score, and they can be counted separately into three different domain subscores , with higher scores indicating more severe voice impairment. In addition, cut-off points have been proposed  to categorize the various scores into either mild, moderate or severe voice impairment [8].The in text citation for the text has been changed fromCM was performed according to the international guidelines [9].toCM was performed according to the international guidelines [10].The in text citation for the text has been changed fromAfter chart review, 19 patients were identified who experienced voice changes after CM. Of these, two made full recovery within fourteen days and were therefore attributed to intubation-related causes [6].toAfter chart review, 19 patients were identified who experienced voice changes after CM. Of these, two made full recovery within fourteen days and were therefore attributed to intubation-related causes [7].The footnotes for the Fig. 2 has been changed fromCut-off scores for categorization of voice impairment severity for the Voice Handicap Index (VHI) [7]toCut-off scores for categorization of voice impairment severity for the Voice Handicap Index (VHI) [8]The in text citation for the text has been changed fromThe 6.3% incidence of voice changes and 3.7% incidence of confirmed RLN injury after CM in our study is greater than described in the known literature, where RLN injury rates of 0–3% have been reported after CM , and even < 1% in larger case series . However, the incidence is dependent on the exact definition of the outcome measure and the intensity of the search for this complication. When routine indirect laryngoscopy was performed after CM in a historical series, Widström found a 6% rate of vocal cord palsy [11], already suggesting this problem might be overlooked during regular clinical follow-up in other reports . Furthermore, our current study is focused on functional patient-reported outcome, with reviewing of both medical and nursing records, which might be a more accurate method to evaluate the magnitude of this problem. The majority of available reports on RLN injury or palsy after CM  rarely provide well-defined data definitions on morbidity outcomes, and it is unclear whether these incidence rates were scored based on functional complaints or laryngoscopy findings, or when the follow-up evaluation was performed.toThe 6.3% incidence of voice changes and 3.7% incidence of confirmed RLN injury after CM in our study is greater than described in the known literature, where RLN injury rates of 0–3% have been reported after CM , and even < 1% in larger case series . However, the incidence is dependent on the exact definition of the outcome measure and the intensity of the search for this complication. When routine indirect laryngoscopy was performed after CM in a historical series, Widström found a 6% rate of vocal cord palsy [4], already suggesting this problem might be overlooked during regular clinical follow-up in other reports . Furthermore, our current study is focused on functional patient-reported outcome, with reviewing of both medical and nursing records, which might be a more accurate method to evaluate the magnitude of this problem. The majority of available reports on RLN injury or palsy after CM  rarely provide well-defined data definitions on morbidity outcomes, and it is unclear whether these incidence rates were scored based on functional complaints or laryngoscopy findings, or when the follow-up evaluation was performed.The in text citation for the text has been changed fromTraditionally, CM remains the diagnostic test with thehighest negative predictive value to rule out mediastinallymph node (N2) disease . In the current guidelineson mediastinal staging for lung cancer patients, confirmatoryCM is still indicated in situations of high clinicalsuspicion of mediastinal metastases if endoscopic stagingprocedures such as endobronchial (EBUS) and esophagealendosonography (EUS) are negative , and is advised to prevent unforeseen N2 disease at surgical resection. However, with the advancements and increasingaccuracy of these endoscopic staging techniques, the role of CM has been questioned .toTraditionally, CM remains the diagnostic test with thehighest negative predictive value to rule out mediastinallymph node (N2) disease . In the current guidelineson mediastinal staging for lung cancer patients, confirmatoryCM is still indicated in situations of high clinicalsuspicion of mediastinal metastases if endoscopic stagingprocedures such as endobronchial (EBUS) and esophagealendosonography (EUS) are negative , and is advised to prevent unforeseen N2 disease at surgical resection. However, with the advancements and increasingaccuracy of these endoscopic staging techniques, the role of CM has been questioned .The original article has been corrected.


Notably, between IVF and ICSI SCM, no significant difference in the concentration of the metabolites was found. Our results suggest that ICSI in non-male factor does not alter the SCM metabolomic signature during 96 hours of embryonic development.Intracytoplasmic sperm injection (ICSI) was developed to overcome male factor infertility, however, there recently has been an increasing trend in ICSI usage irrespective of the etiology, demonstrating an overuse of this insemination technique. There is a limited knowledge on the behaviour of ICSI derived embryos in non-male factor infertility patients. Metabolomic assessment of preimplantation embryos in conjunction with morphological evaluation can provide better understanding of embryonic behaviour. Hence, this study was undertaken to explore if there are any metabolomic differences between IVF and ICSI derived sibling day-5 blastocysts from non-male factor infertility patients. This prospective study included nineteen couples with non-male factor infertility undergoing Assisted Reproductive Technology. The sibling oocytes retrieved from each patient were randomly assigned to two groups and inseminated either by IVF or ICSI. Spent culture media (SCM) in which embryos were cultured up to day 5 were collected and investigated using sensitivity enhanced NMR based metabolite profiling utilizing high resolution (800 MHz) NMR equipped with cryogenically cooled micro-coil (1.7 mm) probe. The metabolomic signature between IVF and ICSI derived sibling blastocysts was assessed. A significant reduction in the concentrations of pyruvate, citrate, glucose and lysine were observed in both IVF and ICSI sibling embryos compared to medium control (P< 0.05–0.001). Further, histidine and valine level was found lower in ICSI embryos compared to medium control (P<0.05) during 96 hours of  Non-male factor infertility conditions constitute approximately half of all infertility cases  and usuaOne reason to use ICSI in normo-responding patients to controlled ovarian stimulation (COS) with non-male factor conditions is due to an irrational fear of total fertilization failure . Higher Apart from the contradictory reports on the effectiveness of ICSI in non–male factor infertility, there are several identified risks associated with the technique such as the injection of polyvinyl pyrrolidone (PVP) exposed sperm, variations in temperature and time during ICSI, mechanical and osmotic trauma associated with the injection and suction, meiotic spindle damage and somatic cell DNA contamination –20. Thouin vitro derived human embryos and found that mode of fertilization does not impair the embryo metabolism in moderate male factor infertility patients [Considering all these facts, there is a need to thoroughly understand the embryonic behavior in relation to the mode of insemination in non-male factor infertile patients. While embryo morphology or morphokinetic evaluation can help in quick selection of embryos, it cannot precisely predict the embryo developmental capacity. Embryo metabolomics is an alternative and sensitive method that provides better insight in metabolic physiology and behavior of human embryos ,32. A repatients . This grin vivo brain metabolomics is performed using small animal models where ultra-sensitive analytical tools are used [The growing field of metabolomics promises immense opportunities for the investigation of cellular metabolism and viability using biological mixtures. It involves quantitative analysis of a large number of small molecules (MW ~1000 Da) in biological specimens in one-step. Application of metabolomics to human embryos has provided alternative, adjunctive technologies to understand the metabolic physiology and behavior ,32,34. Aare used ,40. TherTo the best of our knowledge, this is the first study to investigate metabolomics signature in human embryos utilizing the state-of-the-art non-invasive and highly sensitive NMR technology. Notably, the use of split IVF/ICSI in this study provided the opportunity to assess metabolomics signatures in the sibling embryos that were able to develop to morphologically optimal blastocyst on day 5.2 ii) having regular menstrual cycles without any abnormality/surgery to the reproductive system as disclosed in their medical history; ii) absence of pathological conditions such as endometriosis, tubal abnormalities iii) absence of other metabolic/ endocrine system-associated diseases, such as hypo/hyperthyroidism or hyperprolactinemia iv) the male partners of the patients had semen characteristics above the WHO 2010 reference range [This prospective study included infertile couples (n = 19) undergoing assisted reproductive treatment at the University infertility clinic between April 2019 to January 2020. Institutional Ethics Committee approval  was obtained before the initiation of the study. Patients who agreed to sign the informed consent and fulfilled the following criteria were included in the study: i) women < 35 years of age with body mass index between 18.5–22.9 kg/mce range . The patIn order to assess the DNA fragmentation, the spermatozoa from each male partner were subjected to TUNEL assay as described earlier with minor modifications . Briefly2 for 2–3 h.COS was performed using antagonist protocol. Administration of recombinant FSH  was initiated from Day 2 of the cycle. The starting dose of rFSH was 225 to 450 IU/day depending on age, Antimullerian hormone level and antral follicular count. Thereafter, the rFSH dose was increased/decreased depending on the ovarian response until the day prior to hCG administration. Pituitary down-regulation was performed with daily administration of GnRH antagonist  starting from Day 5 of the stimulation. Oocyte maturation was triggered by the administration of recombinant human chorionic gonadotropin  when at least four follicles reached a mean diameter of 18 mm. Oocytes were collected by transvaginal ultrasound-guided follicular aspiration under anesthesia. Oocyte cumulus complexes (OCC) were rinsed and placed in Onestep medium  at 37°C under 6% CO2 and 5% O2 in MIRI® Multiroom incubator . Post 16-18h co-incubation, cumulus cells and zona bound spermatozoa were removed entirely by mechanical pipetting and repeated washing steps. Sperm survival was assessed from the insemination droplets using inverted microscope. Subsequently, embryos were transferred individually to freshly prepared 30μL droplet of Onestep medium covered with oil. After fertilization check, embryos were cultured at 37°C, 6% CO2 and 5% O2 in MIRI® Multiroom incubator.The sibling OCC’s were randomly assigned to split insemination by conventional IVF and ICSI techniques. Briefly, conventional IVF was performed in 80 μL insemination droplet containing 15,000 to 20,000 spermatozoa from the processed fraction of the ejaculate, overlaid with pre-incubated oil . OCC was transferred to each insemination droplet and co-incubated with spermatozoa at 37°C, 6% CO2 and 5% O2 in MIRI® Multiroom incubator.In ICSI, single sperm was selected under an inverted microscope and injected into the denuded metaphase II oocyte under standard laboratory conditions using Olympus-Narishige workstation. Following injection, oocytes were rinsed and cultured individually in 30 μL droplet of Onestep medium covered with oil. Fertilization assessment was done at 16–18 h post ICSI and embryos were transferred to the freshly prepared 30 μL droplet of Onestep medium and cultured at 37°C, 6% COEmbryos from both the groups were assessed for cell number, developmental progression and morphological abnormalities at a regular interval until day 5 of development as per the ESHRE consensus using inverted phase-contrast microscope . BlastocDNA fragmentation in the processed fraction of the spermatozoa and post co-incubation was assessed by sperm chromatin dispersion (SCD) test with minor modifications . Briefly2O) solution containing a pre-calculated amount of TSP  propionate) as a standard reference compound and transferred to 1.7 mm NMR tubes. Thus, all the metabolites present in the SCM were diluted upto ~1.4 times with the dilution solution. The dilution solvent was prepared by adding 0.05g of TSP/mL D2O and diluting by factor of 10 using D2O solvent. This solution (~10 μL) was added to 25 μL culture medium sample to get a working solution containing 8.29 mM of TSP.The spent culture media (SCM) samples were thawed for 10 min at room temperature. 25 μL of each sample was diluted to 35 μL using deuterium oxide (D1H NMR spectra were obtained using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. CPMG 180 degree pulse train for a duration of 12 ms was used to suppress the protein signals from the media. Each spectrum was obtained using 9615 Hz spectral width, 5s relaxation delay, 16k time domain points, 4 dummy scans and 256 transients.NMR experiments were performed on 800 MHz Bruker Advance NMR spectrometer equipped with 1.7 mm cryo-probe at 298 K. One dimensional (1D) 1H spectra were acquired which included medium control (n = 37) and 47 SCM each from IVF and ICSI group. A total of 12 metabolite peaks were identified based on spectral databases [The time domain data from Free Induction Delay (FID’s) were apodized with a Shifted Sine Bell window function (SSB = 2) and zero-filled to 65536 points prior to Fourier transform. Bruker Topspin version 3.6.2 software was used for NMR data acquisition and processing. A total of 131 1D atabases ,46. RelaThe clinical and demographic characteristics of the participants enrolled in the study has been represented as mean ± standard deviation (mean±SD). A univariate analysis of the embryological characteristics was implemented using GraphPad InStat 3.0 statistical package. Subsequently, a descriptive analysis of the 12 metabolites across IVF versus ICSI was carried out using CRAN R 3.6.1 , where SThis prospective study included 19 infertile couple who underwent ART treatment. Patient demographics and clinical characteristics are depicted in Metabolomic signature from SCM was assessed in a total of 131 1D proton NMR spectra. Spent media from only good quality blastocysts on day 5 of development as per Istanbul consensus  was usedin vitro culture. However, only four metabolites from IVF embryos and six metabolites from ICSI embryos demonstrated significant reduction compared to medium control  did not demonstrate any identifiable differentiation between the metabolite levels of IVF and ICSI derived embryos . This indicates that IVF and ICSI derived embryos are comparable in terms of their metabolite utilization in vitro over a period of 96 hours of development.Subsequently, relative concentrations of SCM metabolites were compared with the media control to understand the utilization of metabolites from the culture medium. A reduction in metabolite levels was observed for all identified metabolites , which i embryos . FurtherPaternal factors have shown to play an important role in embryo development. Though, spermatozoa from only non-male factor patients were used in this study, we made an attempt to look into the impact of specific sperm defects such as head abnormality and DNA fragmentation on SCM metabolites. In the case of sperm head defects, the samples were categorized as patients having <15% (n = 7) and >15% sperm head defects (n = 12). SCM intensities were comparable between IVF and ICSI groups irrespective of sperm head defects . SubsequFurther, metabolite levels were tested to determine any association with sperm DNA fragmentation as measured by the TUNEL assay. Similar to sperm head defects, a comparison was made between the ejaculates having <10% (n = 10) and >10% TUNEL positive sperm (n = 9). It was observed that embryos derived from the ejaculate having <10% DNA fragmented spermatozoa had comparable metabolite levels in both IVF and ICSI group . SimilarExisting evidence do not support the preference for ICSI over IVF in non-male factor patients. However, increase in the number of ICSI cycles across the world, controversial rationale for the increase in the number of ICSI cycles and the risks associated with the technique has prompted us to determine whether any metabolomic differences exist in sibling embryos derived from normozoospermic ejaculates. As ICSI bypasses natural selection process, we hypothesized a differential metabolomic signature in this cohort of sibling embryos. However, our data demonstrated comparable level of metabolites in the good quality sibling blastocysts. These observations imply no overall metabolomic differences in the blastocysts derived from non-male factor infertility partners in relation to the insemination techniques used.in vitro for 96 h under identical laboratory conditions.Though, fertilization rate was higher in ICSI group, the embryo progression and quality during the preimplantation development was comparable between two groups which is in agreement with the earlier study . On the in vitro culture. Specially, only ICSI-derived blastocysts showed reduced consumption of histidine and valine in comparison to medium controls. At the moment it is not possible to explain the significance of this subtle variation, but definitely warrants further investigation in this direction. However, multivariate analyses demonstrated the lack of relationship between the method of fertilization and the levels of metabolites  generated by dead or abnormal spermatozoa may negatively affect the fertilization and subsequent embryonic development. To understand the possible impact of ROS during conventional IVF, we assessed sperm survival soon after the termination of gamete co-incubation. Sperm DNA damage was also assessed prior to the insemination and soon after the termination of sperm-OCC co-incubation. Sperm survival did not vary significantly across the samples. Similarly, sperm DNA damage post-insemination was comparable to pre-insemination level. These observations suggest no significant negative influence of factors during gamete co-incubation on the embryo quality and metabolomic signature.Paternal genetic alterations may affect embryo viability and reproductive outcomes. It has been shown that paternally transmitted DNA lesions influence the metabolism in preimplantation embryos ,56. Low TM enabled fast NMR data acquisition with more than 200-fold reduction in experiment time. This tool is extremely useful for investigation of metabolism, particularly, using mass limited samples such as the low volume SCM samples as used in this study. To the best of our knowledge, this is the first study to investigate metabolomics signature in human embryos utilizing the state-of-the-art non-invasive and highly sensitive NMR technology.A recent study by Leary and Sturmey  analyzedIt has been shown that embryos carrying induced DNA lesions develop normally until blastocyst stage and were morphologically indistinguishable from control embryos . Hence, The use of sensitivity enhanced NMR spectroscopy to compare the SCM metabolomic signature in sibling blastocysts revealed that in non-male factor infertility patients, the ICSI does not significantly impact on the metabolism of embryos. Though, our hypothesis was not proven in this study, the technique should be utilized for the indication it was developed for, and not as a first-line insemination method for patients with healthy sperm and oocyte. This is a comparison between the metabolomic profiles of day-5 IVF and ICSI blastocysts, among which embryo transfer selection is performed. In this pilot study, we are only interested in whether the insemination method affects blastocyst metabolites. Further research should focus on comparison of the metabolomic profiles of day-5 blastocysts that resulted in pregnancy and those that failed to implant.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Nursing homes (NHs) typically focus on health-related quality of life (HRQoL) among residents aged 65 and over despite approximately 7% of NH residents are younger (aged 18-64). Research suggests that the needs of younger NH residents are not being met and they may have low HRQoL. However, differences in HRQoL of younger and older NH residents may not be apparent in studies that use HRQoL measures designed for research with older NH residents. We hypothesized that the younger residents would have lower HRQoL mean scores than the older (aged ≥ 65) residents using a HRQoL measure based on the HRQoL score derived from Resident Assessment Instrument – Minimum Data Set 2.0 items. The measure uses items that emphasize physical aspects of quality of life rather than social aspects. In a sample of 21,129 residents from 94 NHs in Western Canada, we performed descriptive analyses, t-test, chi-square test, and an adjusted propensity score (PS) analysis through retrospective cohort study from years 2016 to 2017. The HRQoL index score ranged from -.351 to .996 . In the PS model, the adjusted mean score for younger was higher than for older adults with a mean difference at 0.061  (p<.001). Other domains such as mental health condition of quality of life must be examined in younger NH residents because it is a crucial factor influencing their daily lives, thereby we can explore a more complete set of HRQoL domains of them and redesign care for their unique needs.


Saccharomyces cerevisiae, including the cell cycle. Two ethanol-responsive lncRNAs in yeast interact with cell cycle proteins, and here, we investigated the role of these RNAs in cell cycle. Our network dynamic modeling showed that higher and lower ethanol-tolerant strains undergo cell cycle arrest in mitosis and G1 phases, respectively, during ethanol stress. The higher population rebound of the lower ethanol-tolerant phenotype after stress relief responds to the late phase arrest. We found that the lncRNA lnc9136 of SEY6210 (a lower ethanol-tolerant strain) induces cells to skip mitosis arrest. Simulating an overexpression of lnc9136 and analyzing CRISPR–Cas9 mutants lacking this lncRNA suggest that lnc9136 induces a regular cell cycle even under ethanol stress, indirectly regulating Swe1p and Clb1/2 by binding to Gin4p and Hsl1p. Notably, lnc10883 of BY4742 (a higher ethanol-tolerant strain) does not prevent G1 arrest in this strain under ethanol stress. However, lnc19883 circumvents DNA and spindle damage checkpoints, maintaining a functional cell cycle by interacting with Mec1p or Bub1p even in the presence of DNA/spindle damage. Overall, we present the first evidence of direct roles for lncRNAs in regulating yeast cell cycle proteins, the dynamics of this system in different ethanol-tolerant phenotypes, and a new yeast cell cycle model.Ethanol alters many subsystems of  Ethanol is a cell stressor in yeast that dampen ethanol production. LncRNAs are RNAs that control many cellular processes. Computational simulations allow us to study the dynamism of cell systems. Therefore, we built a computational model of the yeast cell cycle to investigate how cells respond to ethanol stress. Simulations showed that ethanol stress or spindle damage arrests the cell cycle. Furthermore, the performance of higher and lower ethanol-tolerant strains in poststress recovery growth seems to be related to the cell cycle phase in which cells are stalled. However, two lncRNAs maintain the activity of the cell cycle even in yeast cells under these stresses by repressing specific cell cycle proteins. Finally, this model facilitates analyses of the yeast cell cycle for applied or basic science purposes. Saccharomyces cerevisiae  constrains the node Rad53 to assume only values of ’0’ or ’1’ during simulations. The notation X[Y@n] indicates that the logical functions of the X node will operate such as the Y = ‘n’ level (the X’s node regulator). For instance, Chk1[Mec1@2] will drive the Chk1 node updates assuming Mec1 = 2, although Mec1 will be updated according to its logic equations. This approach enables to peak the maximum level for these nodes but not excluding the possibility to evolve during the simulation. The notation in the equations was designed according to the symbols and rules indicated in the GINsim manual.The numbers in brackets represent the up- or downregulation profiles for each phenotype from the data reported in (PDF)Click here for additional data file.S5 TableThe “-” symbol indicates the lack of differential expression .(PDF)Click here for additional data file.S1 DataName; strain: genomic coordinates start-end; orientation.(PDF)Click here for additional data file.S1 Video(ZIP)Click here for additional data file.


Magnetically responsive composites can impart maneuverability to miniaturized robots. However, collective actuation of these composite robots has rarely been achieved, although conducting cooperative tasks is a promising strategy for accomplishing difficult missions with a single robot. Here, we report multimodal collective swimming of ternary-nanocomposite-based magnetic robots capable of on-demand switching between rectilinear translational swimming and rotational swimming. The nanocomposite robots comprise a stiff yet lightweight carbon nanotube yarn (CNTY) framework surrounded by a magnetic polymer composite, which mimics the hierarchical architecture of musculoskeletal systems, yielding magnetically articulated multiple robots with an agile above-water swimmability (~180 body lengths per second) and modularity. The multiple robots with multimodal swimming facilitate the generation and regulation of vortices, enabling novel vortex-induced transportation of thousands of floating microparticles and heavy semi-submerged cargos. The controllable collective actuation of these biomimetic nanocomposite robots can lead to versatile robotic functions, including microplastic removal, microfluidic vortex control, and transportation of pharmaceuticals. The collective actuation of miniaturized robots is highly desirable for executing cooperative tasks. Won et al. show collective locomotion of ternary-nanocomposite-based magnetic robots that are capable of rectilinear translational and rotational swimming controlled by a pulsed electromagnetic field. Numerous living creatures organize to complete complicated tasks, navigate through harsh environments, and defend against predators. Recently, nature-inspired robotic collectives, e.g. kilobot swarms5 and drone flocks6, have been devised. Multiple mechanical robots equipped with on-board batteries can be organized through sensor-driven communication. Meanwhile, in miniaturized robotics, external-stimuli-responsive particle swarms that can be operated by a magnetic field7, light8, electric field9, and ultrasound10 have been introduced to avoid on-board batteries. In particular, magnetic actuation has attracted considerable attention for developing miniaturized robotic systems owing to the real-time spatiotemporal controllability and high penetrability of external magnetic fields12. Time-varying magnetic fields15 including rotating, oscillating, and precessing offer programmable magnetomotility of microrobots through magnetic torque occurring in a magnetic body. Reconfigurable swarms of magnetic microparticles have been realized by varying the axes and frequencies of the alternating magnetic fields17. However, most studies on collective behavior have focused on colloidal particle systems18. Although particle swarms demonstrate micro-cargo transportation capabilities, they face challenges in the transportation of multitudinous cargos. When the amount of cargo increases, long-distance transport and location control become difficult to achieve in a limited time duration.Collective behavior of active matter is widely prevalent and ranges from cell migration23. For example, polymeric robots in capsule24 or cross25 shape could load and release a miniscule cargo to arbitrary destinations. However, cargo transportation has primarily focused on a single cargo smaller than the size of polymeric robots. Recently, tens of robots have cooperated to carry an object that was larger and heavier than the individual robots26, yet further challenges remain to deliver multiple cargos rapidly and simultaneously. Controllable collective actuation of multiple robots is required to achieve functionalities beyond those offered by single-robot systems.Geometric changes in miniaturized robots are anticipated to improve actuation efficiencies of the robots for cargo transportation. Polymer-composite-based robots are particularly promising since diverse structures, composed of multi-material, can be constructed through polymer processing techniquesIn this study, we presented musculoskeletal-system-mimicking nanocomposite robots capable of agile multimodal above-water swimming. The nanocomposite robots were designed with the aim of generating a water vortex at the air-water interface and transporting multiple cargos via the vortex. Vorticity was controlled by multimodal collective swimming of multiple nanocomposite robots. Thousands of floating microbeads were collected or confined in space through vorticity control by on-demand multimodal collective swimming. Furthermore, the nanocomposite robots transported a heavy semi-submerged cargo that was intentionally prevented from buoying. The multimodal collective swimming of these biomimetic nanocomposite robots demonstrates their potential for overcoming the challenges of limited interactive motions of multiple polymeric robots.27 showing Young’s modulus versus density. We further conceived a magnetic robot with high aspect ratio (AR) and collective actuation of the high-AR robots as a more effective strategy for generating the vortex. When the high-AR robots were magnetically assembled to have large hydrodynamic volume, the magnetically assembled robots were expected to exert a high rotational force and induce a large-magnitude vortex during rotational swimming. Magnetic assembly of the multiple robots required larger magnetic attractive force among robots than drag force to preserve the assembled structure even during rapid swimming. To enhance magnetic attractive forces among robots, we considered a core-shell-like robot by binding highly concentrated magnetic particles only on the robot surface, instead of embedding particles inside the robot since the interparticle magnetic force drastically increases by decreasing interparticle distances29. The robot design that met these requirements was inspired by brachial anatomy  and specific modulus of 46.9 N tex−1  values of the ternary nanocomposites were measured to be 69, 118, and 152 emu g−1, respectively, in accordance with the increased loading of the magnetic particles   was used to normalize these data for enabling comparison with other swimmers, the CNTY robot was found to show an average swimming speed of 180 BL s−1  and AR-6/20-vol.% (yellow triangles) show two intersection points, implying dissipation and recovery of the magnetic attractive force among the soft joints during the assembled rectilinear translational swimming. The influence of dissipative magnetic interactions was confirmed using the deconvoluted harmonic sine functions of the trajectory  thermal imager. For seven 10 vol.% CNTY robots, the rotational mode at 6.7 Hz led to a more significant water vortex than the rectilinear mode at 3.3 Hz  and a surrounding free vortex (thin lines). The reversible transition between the rectilinear and rotational modes enabled the block or transport of 3,350 floating microbeads with a diameter (D) of 255 µm by the 10 vol.% CNTY robots to a desired location  with a weight of 52 mg, which was 82 times heavier than an individual CNTY robot, at 13.3 Hz  using assembled rotational swimming  microstructures41. The biomimetic hierarchical structure of these robots assisted in realizing a stiff yet lightweight long framework, imparting agile above-water swimmability of high-AR CNTY robots and thereby inducing vortices. A vortex-induced transportation strategy was introduced to enable the biomimetic nanocomposite robots to rapidly deliver thousands of miniscule cargos, which is an arduous task for gripping devices. Considering that tethered42 or untethered44 manipulation of miniaturized grippers has been implemented for delicate pick-and-place control of micro-scale objects, the transportation of numerous small objects requires the development of alternative strategies. The CNTY robots designed in this study could rapidly transport and confine thousands of microbeads into specific spaces owing to the multimodal collective swimming of their magnetic modular assembly which facilitated vortex generation and vorticity control. The vortex was intensified by collective swimming of the CNTY robots with increased thickness of the magnetic composite layer; this allowed transportation of millimeter-scale cargos that were 52 times heavier than an individual robot and collection of thousands of microplastics that were scattered at the air-water interface. The collective actuation of biomimetic nanocomposite robots is anticipated to provide versatile robotic applications for microplastic removal46, vortex control in microfluidic platforms47, and transportation of pharmaceuticals49. Moreover, because the magnetic modular assembly of CNTY robots performs on-demand multimodal collective swimming, this proof-of-concept of miniaturized modular robots suggests that more complex robotic tasks can be accomplished through the physical intelligence50 of robot swarms.Collective actuation of CNTY robots shows tremendous potential for expanding the functional scope of carbon-based nanomaterials developed into artificial muscles51. Methane , thiophene , and ferrocene  were used as the carbon source, promoter, and catalyst precursor for MWNT synthesis at 1200 °C, respectively. Synthesized MWNT fibers were constantly drawn into threads in the reactor by passing through a tap-water bath followed by an acetone bath . A thread of MWNT fibers was directly prepared on a roller at a winding speed of 5 m min−1. The 100 fibers of MWNTs were twisted to form a yarn, forming a helical feature and a porous interior between the MWNT fibers.The CNTY was prepared using a floating catalyst chemical vapor deposition methodPDMS prepolymer was mixed with a crosslinking agent  at a 10:1 ratio. Iron particles  were dispersed in the PDMS prepolymer at concentrations ranging from 0 to 40 vol.%. The PDMS–iron-particle mixtures were degassed in a vacuum chamber. Six-centimeter-long CNTY specimens were dip-coated horizontally in the PDMS–iron-particle mixtures for a duration of 1 min. The dip-coated CNTY samples were drained vertically at room temperature for 10 min. The ternary nanocomposites were fully cured in an oven at 130 °C for 3 h, followed by cutting of the resultant specimens into predetermined ARs  for preparing the CNTY robots.−2; UIM-250, UNIX) for 3 min. The heat radiation from the CNTY robots was examined using an FLIR imager . Multiple modular CNTY robots were prepared at ARs of 2.5–6 for the vortex visualization and cargo transport demonstration in Figs. The CNTY robots were actuated at the air–water interface in a container filled with 6.5 mm of water under a pulsed electromagnetic field. The maximum magnitude of the electromagnetic field was measured to be 13 mT. The swimming modes of CNTY robots were changed by switching the magnetic frequency. The swimming modes were analyzed using a high-speed camera , and the swimming trajectories were tracked using the center of mass of each CNTY robot. The vortices were visualized by analyzing the temperature of the water surface after exposing the CNTY robots to NIR light (0.5 W cmSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10


The ciliary body critically contributes to the ocular physiology with multiple responsibilities in the production of aqueous humor, vision accommodation and intraocular immunity. Comparatively little work, however, has revealed the single-cell molecular taxonomy of the human ciliary body required for studying these functionalities. In this study, we report a comprehensive atlas of the cellular and molecular components of human ciliary body as well as their interactions using single-cell RNA sequencing (scRNAseq). Cluster analysis of the transcriptome of 14,563 individual ciliary cells from the eyes of 3 human donors identified 14 distinct cell types, including the ciliary epithelium, smooth muscle, vascular endothelial cell, immune cell and other stromal cell populations. Cell-type discriminative gene markers were also revealed. Unique gene expression patterns essential for ciliary epithelium-mediated aqueous humor inflow and ciliary smooth muscle contractility were identified. Importantly, we discovered the transitional states that probably contribute to the transition of ciliary macrophage into retina microglia and verified no lymphatics in the ciliary body. Moreover, the utilization of CellPhoneDB allowed us to systemically infer cell–cell interactions among diverse ciliary cells including those that potentially participate in the pathogenesis of glaucoma and uveitis. Altogether, these new findings provide insights into the regulation of intraocular pressure, accommodation reflex and immune homeostasis under physiological and pathological conditions.The online version contains supplementary material available at 10.1007/s00018-022-04559-w. It consists of the anterior thicker part called rs plana . Within rs plana .The ciliary epithelium is a bilayer cell alignment comprising the exterior pigmented epithelium (PE) layer and the interior non-pigmented epithelium (NPE) layer. Developmentally, the ciliary epithelium originates from the distal region of the optical cup and is differentiated from two different neuro-epithelial cell origins: NPE and neural retina are derived from the inner layer of the optic cup; and PE and the adjacent retinal pigment epithelium (RPE) are from the outer layer of the optic cup , 3. PrevLike the brain, the eye is one of the few immune privileged sites of the body. Due to the segregated state from the peripheral immune system, the eye contains various myeloid cells that respond to foreign and self-antigens, and locally, tissue-resident macrophages or microglia are the well-characterized immune cell types that mediate baseline immunity in the intraocular tissues –9. ExtenThe ciliary body processes contain many fenestrated capillaries to enable quick exchange of oxygen, fluids and soluble factors that are vital to this multifunctional and interactive tissue , 10. HowThe high-throughput single-cell RNA sequencing (scRNAseq) technology empowers unprecedented depiction of differential cell populations, functionality-related transcriptomic profile, differentiation status and cell–cell communication at single-cell resolution. A number of scRNAseq studies have been conducted to generate cell atlas of various human ocular tissues including the retina, choroid, limbal epithelium and aqueous humor outflow tissues –22. In tScRNAseq was performed on eyes of 3 human donors obtained from Guangdong Eye Bank, Zhongshan Ophthalmic Center, Sun Yat-sen University. The eye pair 1 was from a 41-year-old male donor patient who died due to cerebral hemorrhage. Pair 2 was from a 33-year-old male donor who died from brainstem hemorrhage. Pair 3 was from a 55-year-old male donor whose cause of death was lung cancer. All donors were not diagnosed with ocular diseases. Characteristics of the donors are reported in Suppl. Table 1. Acquisition of the donor eyes for scRNAseq and immunohistological studies were approved by the Medical Ethics Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, and performed in agreement with the tenets of the Declaration of Helsinki.The eyes after corneal excision were immediately immersed in PBS containing 50 U/ml penicillin–streptomycin on ice (please refer to Suppl. Table 1 for detailed information of the elapsed time for the generation of live single cell suspension and of cDNA sequencing libraries after the donor's death) and bisected along the equator so that the anterior and posterior halves were separated. The lens and surrounding zonular fibers were then removed from the anterior half of the eye, followed by cutting the shell of the anterior half into four radial wedges. A microsurgical scissor was used to carefully cut through each wedge along the ora serrata that separates the retina and ciliary body. After removal of the iris and sclera from each wedge, the ciliary body tissue was collected and then subjected to cell dissociation.2  and CaCl2  were added in the digestion buffer, followed by another incubation of the mixture at 37 °C for 15 min with occasional shaking. The digestion was terminated by the addition of heat-inactivated fetal bovine serum  and the tissues were further dissociated by repeated pipetting and gentle mincing and filtered through a 70-μm strainer and then a 40-μm strainer. Cells were rinsed with 0.04% BSA/PBS for 3 times after centrifugation at 1,500 rpm for 5 min and re-suspended with 3 ml 0.04% BSA/PBS.The ciliary body tissues from each pair of the eyes were incubated in 25 ml PBS containing 0.04% BSA, 1.2 U/mL dispase and 50 U/mL penicillin–streptomycin at room temperature for 15 min. Thereafter, trypsin , hyaluronidase , kynurenic acid , MgClFor cell count and viability test, cells were dispersed by gentle pipetting and passed through a 35-μm strainer. A small aliquot of the cell suspension was then mixed with an equal volume of Acridine Orange/Propidium Iodide solution for staining. Cell number and viability were measured by the Countstar automated cell analyzer. Approximately 400,000–800,000 ciliary cells with > 80% viability were obtained from each preparation.A BD Rhapsody analysis system was used to prepare single-cell whole transcriptome. Cell suspension was loaded into the BD Rhapsody cartridge containing a microwell array with > 220,000 partitions. Single cell capture was achieved by random distribution of single cells that settled into the microwells by gravity, followed by the addition of beads with oligonucleotide barcodes for pairing with the cells. After cell lysing, the barcoded beads containing the captured mRNAs were retrieved, washed and then subjected to reverse transcription and Exonuclease I digestion. Single-cell transcriptomic sequencing libraries were prepared by following a series of PCR steps including random priming and extension (RPE), RPE PCR and whole-transcriptome amplification (WTA) index PCR according to the manufacture’s instruction. Normalized libraries were sequenced on NovaSeq Illumina with a 150 bp paired-end run.fastp preprocessor tool with default parameters [Quality control and preprocessing of the FASTQ sequencing data was performed using the rameters . The UMIrameters  that modrameters . A Seurarameters  were filp < 0.01; and pct.1 > 10%. Alternatively, to identify potential marker genes of both PE and NPE populations, the FindMarkers function was employed via comparing each PE/NPE population with the group of all other non-epithelial ciliary cells using the following threshold metrics: Log2FoldChange > 0.25; one-sided Wilcoxon rank sum test p < 0.05; pct.1 (in PE/NPE) > 25%; and pct.2  < 5%), and the resulted 40 common genes for both PE and NPE was obtained (refer to Suppl. Table 4).Cell clusters were acquired via the graph-based clustering approach in Seurat. The FindNeighbors input dimensions of reduction was set to 40 which was determined by principal component analysis (PCA) via the RunPCA function, and the Findclusters resolution parameter was set to 0.1. The projected clusters were visualized after nonlinear dimensional reduction by Uniform Manifold Approximation and Projection (UMAP) and annotated using known marker genes. A top 50 ranked list of predicted markers  for each cell cluster were also identified by the FindAllMarkers function according to the following threshold metrics: Log2FoldChange > 0.25; one-sided Wilcoxon rank sum test Subclusters in the SMC, endothelial cell and macrophage populations were determined through the aforementioned normalization, regression and graph-based clustering pipeline with differential PCA and resolution parameters and annotated based on expression pattern of previously published gene markers (refer to Suppl. Table 6 for gene abbreviation list) as visualized on UMAP or examined by immunofluorescent staining.Additionally, monocytes and macrophages in human ciliary body as well as the microglial cell clusters from human retinas, which are also resident mononuclear phagocytes, were subjected to clustering analysis. We obtained three sets of raw counts data of the microglial cell clusters from existing retinal scRNAseq datasets –22 and cp < 0.05; and pct > 10%) between two relevant clusters/subclusters. KEGG functional enrichment analysis was performed on the DEGs using a hyper-geometrical statistical test with p < 0.05 considered statistically significant. Gene set enrichment analysis (GSEA) was also employed using the indicated GO pathways (Suppl. Table 7).Finally, the FindMarkers function was implemented to obtain differentially expressed genes  tool which incorporates a repository of 501 secreted proteins (ligands) and 585 membrane proteins (receptors) . These pTg-Prox1-EGFP transgenic mice . An EGFP-BGHPolyA-FRT expression cassette was introduced by recombineering at the start codon (ATG) of Prox1 gene in bacterial artificial chromosome (BAC) clone RP23-385H16 that contains an approximately 215 kb mouse Prox1 genomic DNA (including the entire Prox1 gene and the 134 kb sequence upstream of the start codon and 41.9 kb sequence downstream of the stop codon). Following microinjection of the transgenic construct into mouse donor zygotes, the resulting founder Tg-Prox1-EGFP BAC mice were propagated on a C57BL/6J background. Genotyping was performed by detecting the Prox1-EGFP transgene using the following primer pair:To visualize Prox1 + lymphatic vessels in vivo, we generated 5′-CCATCTTAAACCAGTGGAGCAGA-3′5′-AACTTGTGGCCGTTTACGTCG-3′Faithful lymphatic expression of the Prox1-EGFP transgene was confirmed in LYVE1 stained limbus lymphatic vessels and other lymphatic vascular beds.The Institutional Animal Care and Use Committee (IACUC) of Zhongshan Ophthalmic Center, Sun Yat-sen University has approved the mouse studies. All animal experiments were in accordance with Use of Animals in Ophthalmology and Vision Research.Human donor eyes were fixed in 4% paraformaldehyde (PFA)/PBS overnight, infiltrated with 30% sucrose for 48 h, embedded in OCT medium and processed for cryosectioning and standard histological examinations. Cross-Sects. (10 μm in thickness) of the eye were blocked with PBS containing 0.1% Triton X-100 and 5% donkey serum for 1 h and incubated with primary antibodies at 4 °C overnight and fluorescently labeled secondary antibodies and DAPI at room temperature for 45 min, with adequate PBS washes after each incubation. After mounting, the sections were imaged under a Nikon CS2 confocal microscope.Tg-Prox1-EGFP transgenic mice and fixed in 4% PFA/PBS overnight. The limbus and ciliary body tissues were dissected from the fixed eyes under a stereomicroscope, followed by blocking in a blocking buffer  and incubations with anti-LYVE1 antibodies (diluted in blocking buffer) for two days at 4 °C and appropriate secondary antibodies at 4 °C overnight. After washing, the immune stained tissues were mounted using VECTASHIELD mounting medium containing DAPI and photographed with a Nikon CS2 confocal microscope.For tissue whole mount staining, eyes were harvested from postnatal day 21 (P21) pars plicata and pars plana regions  without adjacent ocular tissues were carefully dissected and dispersed into single cell suspension as described in Methods. Individual cells were captured using the BD Rhapsody platform and then subjected to scRNAseq analysis. A total of 20,590 individual ciliary cells from the three scRNAseq experiments were initially obtained and integrated into a single matrix dataset (Suppl. Table 2). After filtering out rare reads, low-quality cells and presumed doublets (see Methods), single-cell transcriptome of a total of 14,563 individual ciliary cells were retained, with each sequencing sample ranging from 3848 to 5370 cells (Suppl. Table 2). The average genes per cell was 1778 and the total gene number reached 16,088, in which 15,852 genes were common genes detected in all three samples .To determine the precise cell constitution in the human ciliary body, we performed scRNAseq experiments on the ciliary body tissue from 3 pairs of human donor eyes, with each eye pair used as a sequencing sample (Suppl. Table 1). The pieces of ciliary body tissue including the We then sorted the cells into 14 individual clusters, as visualized by UMAP, based on their gene expression profiles and annotated the clusters using known marker genes  and NPEp < 0.05) between these two cell types , indicating similar transcriptomic features of the ciliary epithelium bilayer. In agreement with a previous study [Next, we compared PE and NPE transcriptome and found only a small proportion of genes  were differentially expressed  identified two populations of smooth muscle cells in human ciliary body: one represents vascular smooth muscle cell (VSMC) that covers ERG + blood ECs; the other one is nonvascular ciliary muscle cell (CMC) Fig. A, B. WitMacrophage was one of the greatest contributors to the cell constituents in our scRNAseq analysis. Subclustering analysis on the entire macrophage population was able to segregate this cell population into two transcriptionally distinct subclusters Fig. A. AnalysNext, we combined our monocyte/macrophage data with the raw counts of microglia clusters from existing human retinal scRNAseq datasets –22 to prProper functioning of the ciliary body requires cooperative signaling arrangements of the epithelium, smooth muscle, immune cells and other stromal cells. We next focused our attention on ligand-receptor pairs among major ciliary cell types to infer potential intercellular communications in this tissue. Through CellPhoneDB (see Methods), we revealed that genes involved in a total of 1046 ligand-receptor pairs (1396 as the input repository) were present in our scRNAseq dataset. Based on the transcriptional expression levels of the ligand and receptor genes and the percentage of cells expressing these genes, we computed interaction scores of any possible ligand-receptor pairs between two cell types. After statistically analysis of the scores using a one-sided Wilcoxon rank-sum test, we identified 8,079 cell–cell interactions formed between homotypic or heterotypic cell pairs across the 14 human ciliary cell types , and EGF, TGFβ, DLL4/Jagged, VEGF and FGF were among the most common growth factor family members that interact with their receptors in human ciliary body . As revealed by the cell communication analysis, interactions between EGF family members and their receptors were majorly formed among NPE, PE, Schwann, EC, fibroblast and monocyte populations, suggesting EGF pathways may be commonly required for functioning of many ciliary cells Fig. E. TGFβ aCells that reside in the ciliary body play key roles in the ocular physiology as they constitute the anterior portion of the uveal tract and are responsible for the production of aqueous humor and vision accommodation. In this study, we generated a cell atlas for the ciliary body in humans using scRNAseq technology. Cluster analysis identified 14 distinct cell types, representing the epithelial, vascular, neural, stomal and immune cell populations of human ciliary body. We revealed cell-type discriminative gene markers or expression patterns and systemically inferred cell–cell interactions across the ciliary body tissue including those potentially involved in the pathogenesis of glaucoma and uveitis. Although a previous study has profiled the single-cell transcriptomics of ciliary cells in combination with the adjacent neural retina cells in the mouse eye , we provIn comparing the cellular constituents in human ciliary body with those in mouse ciliary body and the contiguous retina tissue , we founRax gene led to ectopic formation of the retina tissue and hyperproliferation of RPEs [RAX is a homeobox-containing transcription factor highly expressed in retinal progenitor cells , 52. RAX of RPEs . As NPE  of RPEs , it is u of RPEs  and LHX2 of RPEs  in the aDespite the common progenitor cells during early eye development, PE and NPE undergo distinct differentiation steps at the late embryonic stage  and evenThe secreted aqueous humor can be drained through both the conventional outflow pathway consisting of the trabecular meshwork and Schlemm’s cancel and the unconventional uveoscleral pathway involving interstitial spaces of the ciliary muscle . The absThe ciliary muscle controls lens accommodation to focus on near objects and facilitates the draining of aqueous humor into Schlemm's canal, and has been used as the therapeutic target to treat cycloplegia  as well One of the major cell types identified was the macrophage population, supporting the notion that cells of the myeloid lineage primarily make up the immune repertoire of the healthy uveal tract . The cilThe CellPhoneDB computational approach allowed for the identification of potential cell–cell interactions in human ciliary body microenvironment. The demonstration of collagen as the most common ligand involved in ECM-integrin interactions, despite that the ciliary body is renowned as the origin of zonule fibers that are primarily made up of fibrillin , predictIn summary, our study has revealed the molecular taxonomy that controls the epithelium and smooth muscle functionality, the transitional states of resident macrophage and the cellular communication in human ciliary body at single-cell resolution, thus bringing new understanding to the regulation of intraocular fluid and immune homeostasis under physiological and pathological conditions.When working with scRNAseq samples from postmortem human tissues, a significant limitation is the exceeding lability of the RNA molecules. Although a previous study has documented that the death-to-preservation time lag (2–6 h) indeed resulted in lower RNA integrity of the ciliary body tissue, longer postmortem time  did not appear to further impair the RNA integrity in human ciliary body . In our Supplementary file1 (PDF 4774 KB)Supplementary file2 (XLSX 12 KB)Supplementary file3 (XLSX 12 KB)Supplementary file4 (XLSX 50 KB)Supplementary file5 (XLSX 22 KB)Supplementary file6 (XLSX 27 KB)Supplementary file7 (XLSX 15 KB)Supplementary file8 (XLSX 11 KB)Below is the link to the electronic supplementary material.


Small RNAs (sRNAs) are an important group of non-coding RNAs that have great potential as diagnostic and prognostic biomarkers for treatment of a wide variety of diseases. The portability and affordability of nanopore sequencing technology makes it ideal for point of care and low resource settings. Currently sRNAs can’t be reliably sequenced on the nanopore platform due to the short size of sRNAs and high error rate of the nanopore sequencer. Here, we developed a highly efficient nanopore-based sequencing strategy for sRNAs (SR-Cat-Seq) in which sRNAs are ligated to an adapter, circularized, and undergo rolling circle reverse transcription to generate concatemeric cDNA. After sequencing, the resulting tandem repeat sequences within the individual cDNA can be aligned to generate highly accurate consensus sequences. We compared our sequencing strategy with other sRNA sequencing methods on a short-read sequencing platform and demonstrated that SR-Cat-Seq can obtain low bias and highly accurate sRNA transcriptomes. Therefore, our method could enable nanopore sequencing for sRNA-based diagnostics and other applications. Small RNAs (sRNAs) are a diverse class of non-coding RNA that include microRNA (miRNA), small nucleolar RNA (snoRNA), tRNA fragments (tRNAf) and piwi-interacting RNA (piRNA) among others. They are important in a variety of cellular functions, including development and regulation of gene expression . FurtherNanopore sequencers, in particular, the commercially available sequencers from Oxford Nanopore Technologies (ONT), offer many advantages for diagnostic applications, including low costs for equipment and consumables. Unlike other sequencing technologies, where data are analyzed after collection, nanopore devices are capable of real time analysis and on-chip target enrichment , making Previous approaches to detect sRNAs on ONT devices involve using chemical modifications to add bulky residues to slow the voltage driven translocation of molecules through the pore and increase the signal, however it is not possible to achieve whole transcriptome sequencing using this method . In anotAll previous approaches using rolling circle amplification to aide in nanopore sequencing have used phi29 DNA polymerase due to its high processivity and strong strand displacement. We reasoned that a highly strand displacing reverse transcriptase (RT) could achieve the same goal directly on RNA templates and would therefore allow for direct amplification of sRNA. One candidate group of enzymes to perform such a function are Group II intron reverse transcriptases. Group II introns are retrotransposons that encode a self-splicing ribozyme as well as a reverse transcriptase. To complete the retrotransposition cycle, the intron-encoded reverse transcriptase must accurately copy its cognate ribozyme which is large and highly structured. While homologous to the retroviral encoded reverse transcriptases typically used in biotechnology, this selection pressure has led to the evolution of high processivity, high fidelity and strong strand displacement activity in the Group II intron RTs .In this paper we show that it is possible to perform rolling circle reverse transcription using Group II intron RTs, with much better performance than retroviral RTs. We developed an efficient sequencing strategy that allows for adapter ligation, circularization and rolling circle reverse transcription of cellular sRNAs without intermediate purifications. The resulting cDNA products can be used directly or further amplified before being sequenced on the ONT MinION device. Finally, we show that our method for converting sRNAs into concatemeric cDNAs (SR-Cat-Seq) can be used to reconstruct high-fidelity and low-bias sRNA transcriptomes from error-prone nanopore data.Circular RNA was prepared using 10 replicates of 20 μl reactions each containing 50 pmol of a 42nt RNA oligo , oligo 1The following reverse transcriptases (RTs) were tested: Avian Myeloblastosis Virus RT , Moloney Murine Leukemia Virus RT , ProtoScript II RT , SuperScript IV RT , WarmStart RTx , Thermostable Group II Intron RT  and Induro RT (NEB M0681). Each reverse transcription reaction was performed under the manufacture’s recommended buffer conditions with 1 μl of the stock concentration of the enzyme. Each reaction contained 1 pmol of either circular or linear RNA oligo , oligo 1The top RNA strand of the adapter , oligo 3For RNA oligos or the miRXplore synthetic reference miRNAs, 5 pmol of RNA was used as input. For human brain RNA, sRNAs (< 200 base pairs) were isolated from 10 μg of total brain RNA using RNA XP beads (Beckman Coulter) following the manufacturer’s protocol. 50 ng of isolated sRNA was used as input. For the SR-Cat-seq workflow, the RNA was first denatured by heating to 70°C for 2 minutes and placed on ice immediately. The ligation mixture was then added to the RNA. The ligation mixture contained 1x T4 RNA ligase buffer (NEB M0204), 20% PEG-8000, 0.05% Tween-20, 20 pmol of the annealed splintadapter (see above) and 200 units of T4 RNA Ligase 2 Truncated K/Q (NEB M0373) in a total volume of 20 μl. The ligation reaction was incubated at 25°C for 1 hour. Subsequently, 2 units of USER enzyme (NEB M5505) and 4 units of DNase I (NEB M0303) were added to the reaction mixture and incubated at 37°C for 30 minutes to remove the bottom DNA splint strand of the adapter. After the degradation of DNA strand, only RNA is subject to the following circularization step. The T4 RNA Ligase 2 Truncated K/Q was then heat inactivated at 75°C for 5 minutes and then cooled down to 4°C. The reaction mixture was then diluted to a total volume of 40 μL with 1x T4 RNA ligase buffer, 1 mM ATP, 10 units of T4 Polynucleotide Kinase (NEB M0201) and 30 units of T4 RNA ligase 1 (NEB M0437M). The circularization reaction was then allowed to proceed for 1 hr at 25°C. Linear RNAs were then degraded by adding the following mixture of enzymes 2uL XRN-1 (NEB M0338), 1uL 5’deadenylase (NEB M0331), RNase R (purified in house), ATP and Poly(A) Polymerase (NEB M0276L). The reaction mixture was then diluted to 80 μL with 2x Induro RT buffer , 1 μM primer , oligo 2Second strand synthesis was performed using Taq DNA polymerase. A primer , oligo 5Amplification was performed using multiple strand displacement amplification with forward and reverse primers designed to anneal to non-overlapping portions of the adapter sequence (https://www.ncbi.nlm.nih.gov/sra) under project accession number: PRJNA603337. qPCR data was previously published and available in supplementary materials [Short read sequencing data were previously published  and avaiaterials .Adapter ligation and circularization reactions were carried out as described above using a 21 bp randomized RNA substrate with an internal FAM label , oligo 8https://www.R-project.org/) were used to parse the resulting genbank files from the SPADE output to collect the consensus sequences. Consensus sequences generated in this manner could have any random circular orientation, therefore, to phase them we generated all possible rotations of each consensus sequence and aligned the adapter to them using pairwise alignments with the Needleman-Wunsch algorithm as implemented in the R package Biostrings  We chose the first rotation of the sequence that gave the longest un-gapped alignment anchored to either the start or end of the read. The adapter sequence was then trimmed from the rotated consensus sequences to yield the final trimmed consensus sequences.Reads were filtered by length (> 1000 bp) and average quality (> = 7) and then converted to FASTA format. SPADE  was usedhttps://bioconductor.org/packages/Biostrings.) We then determined the edit distance between the consensus sequence and the reference including gaps, insertions, and mismatches. The edit distance was subtracted from the number of aligned bases to determine the percent identity to the reference sequence.We assessed consensus sequence accuracy using a synthetic 21 nt oligo with the sequence of human miRNA hsa-let7a , oligo 1MiRXplore libraries were mapped to the miRXplore reference using bbmap and read counts were generated during mapping. Human RNA libraries were mapped to the human genome (GRCh38) using bowtie  with a sConsidering the 962 miRNAs in miRXplore were present in equimolar amounts, an expected read count was generated by dividing the total number of mapped reads for each library by 962. Reads were then normalized by dividing the raw read counts by the expected read counts. An miRNA represented with the exact expected read count would have a normalized value of 1; over- and under-represented sequences would have values greater and lower than 1, respectively.in silico designed RT with RNase H activity). Two other tested RTs were derived from Group II introns, including thermostable Group II intron RT (TGIRT) and Induro RT. We performed reverse transcription with each RT under the manufacture’s recommended reaction conditions with either a linear or a circularized RNA template. Both linear and circular RNA templates are 42 nt long and share the same sequence. Reaction products were treated with RNase H and RNase If to remove the RNA template and visualized using both agarose gel electrophoresis (for large cDNA products) and denaturing polyacrylamide gel electrophoresis (for short cDNA products). Retroviral RT reactions were allowed to continue for one hour while Group II intron RT reactions were stopped at 15 minutes, because pilot experiments showed that a full hour incubation with the Group II intron RTs would create cDNA products too large to be resolved by the gels. When comparing different RT products resolved by agarose gel electrophoresis, we found that the Group II intron RTs generated larger cDNA products with the circular RNA template, indicating robust rolling circle reverse transcription , Moloney Murine Leukemia Virus RT (MMLV), ProtoScript II RT , SuperScript IV RT  and WarmStart RTx . Subsequently, the DNA splint strand of the adapter is enzymatically degraded. The ligated small RNAs are then diluted to favor intramolecular ligation and circularized with the addition of T4 RNA ligase, followed by a cocktail of exoribonucleases to degrade any remaining linear RNA. The reactions were then diluted a second time to accommodate the buffer requirement for the next step of rolling circle reverse transcription, which was performed in the same tube with addition of Induro RT . The resTo assess the efficiency of RNA circularization in the SR-Cat-Seq workflow, we used a 21-mer, FAM labeled, degenerate RNA oligo , oligo 8To assess the sequencing accuracy of the SR-Cat-Seq workflow, we sequenced a 42-mer RNA oligo, containing the sequence of a human miRNA (hsa-let7a) on the 5’ end and our adapter sequence on the 3’ end, mimicking the ligation product in the first step of the workflow with a fully known sequence. This oligo was circularized and rolling circle reverse transcription was performed using our optimized workflow. The resulting concatemeric cDNA underwent either second strand synthesis (SR-Cat) or amplification by phi29 DNA polymerase (SR-Cat-Amp). We sequenced the libraries and applied the SPADE algorithm  to identTo evaluate the sequencing bias of the SR-Cat-Seq workflow, we sequenced a control set of RNA oligos called miRXplore. MiRXplore is a pool of 962 synthetic miRNAs which are mixed at equimolar concentration and commonly used to benchmark bias in small RNA sequencing workflows. To quantify the sequencing bias, we normalized the read counts such that reads represented in the library at their expected values would have a normalized count of 1, while over-represented reads had a normalized count > 1 and under-represented reads had a normalized count < 1 . The biaWe then applied the workflow to sequence small RNA in human brain samples. The same brain RNA was sequenced with 2 technical replicates for both the SR-Cat and SR-Cat-Amp workflows. We detected various sRNAs such as miRNA, piRNA and tRNA as well as some long non-coding RNA and ribosomal RNA . Both SRIn this work we show that Group II intron RTs have strong strand displacement activity and are capable of quickly producing large concatemeric cDNAs from circular RNA templates. Based on this activity we developed SR-Cat-Seq, a highly efficient one-pot workflow that enables sRNA sequencing on the nanopore platform. The whole workflow includes adding adapters to sRNAs, circularization and rolling circle reverse transcription to produce concatemeric cDNAs from small RNAs. We showed that SR-Cat (without amplification) workflow has minimal sequencing bias, which is comparable to the Low-Bias workflow. However, the amplification step in the SR-Cat-Amp introduced bias to the whole workflow, which remains to be improved. Workflows with low sequencing bias are important in applications such as liquid biopsy where accurate abundance measurements can help to rank targets and guide real-time treatment decisions. Finally, we applied the workflow to sequence small RNAs from human brain RNA and demonstrated similar accuracy and sensitivity to Illumina’s TruSeq, the standard short-read method. In addition, we developed a bioinformatics workflow that can reconstruct highly accurate consensus sequences from the concatemeric cDNA products. High fidelity sequencing is extremely important for small RNA research. Because of the small size of small RNAs, even single mismatch may make mapping difficult and cause incorrect counts. Furthermore, for liquid biopsy applications, tumor specific isomirs are of great interest and high-fidelity sequencing is required for detection.We tested versions of our workflow with and without amplification and found that there were advantages and disadvantages to each. The unamplified workflow (SR-Cat) had the lowest bias and would therefore be the best option to use if an accurate representation of the sRNA transcriptome was desired. However, it also had disadvantages including high input requirements of at least 5 pmol for oligonucleotides or 50 ng of sRNA purified from total RNA. These input requirements would preclude the use of this method for most patient samples without further development. Furthermore, the unamplified workflow had a much higher fouling rate of the nanopores which limited the overall output from each flowcell . While sin situ sequencing [The ability of Group II Intron RTs to perform rolling circle amplification has many possible applications beyond the one explored in this work. Analogous activity in DNA polymerases, such as phi29 DNA polymerase, has enabled many different technologies such as whole genome amplification [quencing  among maS1 Fig(TIF)Click here for additional data file.S2 FigCumulative number of reads sequenced is shown on the y-axis vs time in hours on the x-axis, each line represents an individual MinION flow cell.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.