Diabetes mellitus (DM) remains a global concern in both human and veterinary medicine. Type I DM requires prolonged and consistent exogenous insulin administration to address hyperglycemia, which can increase the risk of diabetes complications such as retinopathy, nephropathy, neuropathy, and heart disorders. Cell-based therapies have been successful in human medicine using the Edmonton protocol. These therapies help maintain the production of endogenous insulin and stabilize blood glucose levels and may possibly be adapted to veterinary clinical practice. The limited number of cadaveric pancreas donors and the long-term use of immunosuppressive agents are the main obstacles for this protocol. Over the past decade, the development of potential therapies for DM has mainly focused on the generation of effective insulin-producing cells (IPCs) from various sources of stem cells that can be transplanted into the body. Another successful application of stem cells in type I DM therapies is transplanting generated IPCs. Encapsulation can be an alternative strategy to protect IPCs from rejection by the body due to their immunoisolation properties. This review summarizes current concepts of IPCs and encapsulation technology for veterinary clinical application and proposes a potential stem-cell-based platform for veterinary diabetic regenerative therapy. Diabetes mellitus (DM), a chronic metabolic disease, is caused by the pancreas' inability to produce/secrete insulin or the inability of the body to use insulin effectively. There are two main types of DM in the veterinary field. Type I DM, formerly classified as insulin-dependent diabetes, is associated with immunological disorders. Defects in pancreatic islet cells are mediated by autoimmune disorders, resulting in the absence of insulin. Type II DM, formerly classified as non-insulin-dependent diabetes, is caused by ineffective pancreatic insulin secretion and/or insulin usage by the body . Type I *009-DQA1*001-DQB1*008 (DRB1*009), DLA-DRB1*015-DQA1*006-DQB1*0.23 (DRB1*015), and DLA-DRB1*002-DQA1*009-DQB1*001 (DRB1*002)  , 14. Anoxpressed , 12. TabAlthough DM is caused by complex factors, this disease can still be treated. Currently, in the veterinary field, therapy management of DM aims to control blood glucose under the renal threshold for at least a 24-h period and prevent the occurrence of hypoglycemia. The American Animal Hospital Association (AAHA) recommends a combination of insulin product, non-insulin therapeutic agents , and dietary management to treat DM. As in human medicine, exogenous insulin preparations can be obtained from porcine insulin zinc suspension (intermediate acting) and recombinant DNA-originated human insulin (long-acting). However, the successful therapy of DM in the veterinary field requires commitment and effort by the veterinarian, patient, and owner, which is the main challenge. Moreover, an effective monitoring system to maintain stable blood glucose is also needed, which poses another challenge . Table 2Stem-cell-based therapies are growing rapidly in the veterinary field. However, stem-cell-based therapies in the veterinary field are mainly used for curing musculoskeletal injuries in sport and companion animals , 24. TheThe ethical issues are still hampering the application of regenerative medicine in the veterinary field. Legalization of manufacturing standard operational procedure (SOP), marketing requirements for cell-based veterinary pharmaceuticals, and veterinary medical prescription requirements for cell-based veterinary pharmaceutical products are not well-developed. A genetic engineering approach that might be used for the production of cell-based veterinary pharmaceutical product is one of the additional obstacles for its translation in veterinary clinics. Pharmaceutical products containing genetically modified organisms (GMOs) have not been authorized under pharmaceutical law . AccordiN-acetyl cysteine to reduce senescence in adult stem cells , but many researchers have shown that adult stem cells have the ability to differentiate toward various types of cells –31. Thisem cells .Oct4, Sox2, c-Myc, and KLF4, while Thomson factors consist of Oct4, Sox2, NANOG, and LIN28 (iPSCs are reprogrammed adult (somatic) cells that have similar characteristics as ESCs. iPSCs were developed in response to the controversial issues of ESCs . Currentnd LIN28 . Even thnd LIN28 . A studynd LIN28 .Stem-cell-based therapy for tissue regeneration is mainly aimed to replace damaged cells that cause many various diseases such as congenital disorders –48, tissin vitro differentiation of MSCs. A comparative study of chemical induction between BM-MSCs and adipose tissue-derived mesenchymal stem cell (AT-MSC) differentiation toward IPCs showed no difference in terms of gene expression level, C-peptide, and insulin production   , collagene (PLL) . The spene (PLL) . Therefone (PLL) . Figure A challenge for mammalian cell encapsulation is choosing the appropriate materials to form a suitable environment for the cells to promote the survival rate and maintain the phenotype of the cells . AlginatThe rapid development of successful encapsulated cell transplantation in animal models can provide a basic concept for translational studies for clinical applications in human medicine. This was pioneered in a translational study of cell encapsulation therapy for DM . Cell enInitially, cell encapsulation was studied to provide immunoisolation to cells that produce specific proteins with therapeutic potential to cure diseases. At present, mammalian cell encapsulation has been used to regenerate tissues or organs . There h+ cells, thereby avoiding transplant rejection (In the past decade, there have been many studies on stem-cell-derived IPCs as an alternative to the use of cadaveric pancreatic islet transplantation . Today, ejection , 165 andejection . For a sejection , 168. Anejection . Moreoveejection .in vitro study of alginate encapsulation in terms of immunological reaction has also been done. In that study, alginate-encapsulated rat MSCs, co-cultured with lymphocytes, induced less secretion of IL-2 (Alginate has been widely used in biomedical science and in regenerative medicine. The first study of alginate microencapsulation was done in 1980, but the results were not satisfactory due to poor biocompatibility . In 1997 of IL-2 . A study of IL-2 , enhance of IL-2 , and mai of IL-2 . Another of IL-2 . Double- of IL-2 , 178.Although many studies have been conducted on manipulating the rejection of IPCs, some studies have aimed to cure DM in humans. The utilization of animal models can have a beneficial effect in the veterinary field, since the findings of these studies can be adapted to treat animal DM. The latest study of IPC encapsulation that involved an animal model reported that macroencapsulation using a specific device called “TheraCyte” successfully provided immunoisolation for hBM-MSC-derived IPC transplantation in mongrel dogs; blood glucose level was maintained in the physiological state for the long term . Based oUntil present, there is no effective strategy to treat DM in the veterinary field. Researchers have studied the strategy of generating IPCs and encapsulation technology in both humans and various animal species. The results can be adapted into veterinary clinical applications. MSCs as adult stem cells have been widely used in clinical application in humans, and therefore, they seem to be a good candidate for generating IPCs. Generated IPCs from MSCs can be an alternative to pancreatic islet donors. The ability of MSCs to modulate the immune system allows successful non-autogenic transplantation. Combining this with the promising strategy of encapsulation technology can enhance the viability of IPCs by providing an immunoisolation barrier to avoid graft rejection. Furthermore, the negative effects of prolonged administration of immunosuppressive agents can be eliminated.Consequently, this strategy can overcome many obstacles to cell transplantation, and this platform might be suitable for allogeneic or xenogeneic cell transplantation in the future. The IPC encapsulation platform can be a promising strategy for DM stem-cell-based therapy in veterinary practice. To fulfill this concept, further studies should be conducted to find an effective strategy for generating the IPCs, select a suitable material, improve encapsulation, and investigate the cell behavior after encapsulation. These studies should be followed by clinical trials in animals to evaluate the functional effect and safety of encapsulation.SK reviewed the literature and wrote the manuscript. SS and KK wrote and edited the manuscript. CS conceived the presented idea, wrote and edited the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Myriapods were, together with arachnids, the earliest animals to occupy terrestrial ecosystems, by at least the Silurian. The origin of myriapods and their land colonization have long remained puzzling until euthycarcinoids, an extinct group of aquatic arthropods considered amphibious, were shown to be stem-group myriapods, extending the lineage to the Cambrian and evidencing a marine-to-terrestrial transition. Although possible respiratory structures comparable to the air-breathing tracheal system of myriapods are visible in several euthycarcinoids, little is known about the mechanism by which they respired. Here, we describe a new euthycarcinoid from Upper Devonian alluvio-lagoonal deposits of Belgium. Synchrotron-based elemental X-ray analyses were used to extract all available information from the only known specimen. Sulfur X-ray fluorescence (XRF) mapping and spectroscopy unveil sulfate evaporation stains, spread over the entire slab, suggestive of a very shallow-water to the terrestrial environment prior to burial consistent with an amphibious lifestyle. Trace metal XRF mapping reveals a pair of ventral spherical cavities or chambers on the second post-abdominal segment that do not compare to any known feature in aquatic arthropods, but might well play a part in air-breathing. Our data provide additional support for amphibious lifestyle in euthycarcinoids and show that different respiratory strategies were used during the marine-to-terrestrial transition in the myriapod lineage. Zn sigg, electronic supplementary material, figure S2). XANES spectroscopy at the S K-edge indicates that S is mostly present as sulfates (figure 2h). Traces of organic sulfur, namely cysteine thiol/thioether/heterocyclic S (2472.2–2473.5 eV) and methionine sulfoxide (2475.7 eV), are shown by XANES to be present in several places of the fossil cuticle, most probably representing remnants and/or breakdown products of the chitin-protein complex . Strikingly, the S map reveals bubble-like subspherical to subcylindrical patches enriched in sulfates at their periphery and depleted at their centre, absolutely invisible with regular light (figure 2g) and closely resembling evaporation stains (‘coffee ring’ effect). These stains are present on the entire surface of the sample (both sediment and specimen) but not enriched within fractures (i.e. not secondary infilling), suggesting very shallow-water to subaerial conditions prior to burial.In addition to transition metals, the fossil contains S  is particularly interesting as its information depth on the order of a couple of hundred micrometres allows unveiling additional anatomical details buried under surface layers (b–e). An elongated and annulated structure originating in the ACT and directed forward very likely represents the left antenna . Anteriorly to the PCT, a trapezoidal to triangular anteriorly oriented structure, richer in As than the rest of the fossil, stands out as a thicker, or more sclerotized, plate, covering posteriorly two triangular, laterally rounded, processes . Racheboeuf et al. )y Laurie . Togethenplatten .E. potii gen. et sp. nov., from a specimen preserved fully articulated (i.e. most likely with no or limited transport) in very shallow-water to subaerial sediments, provides additional evidence for an amphibious lifestyle for euthycarcinoids. A second independent evolution  of a system similar to the book lungs within Arachnida [The recognition of air-breathing adaptation in rachnida , or to trachnida . Our fin


Purpose: To safely care for their newborn patients, health-care professionals (HCP) must undergo frequent training to improve and maintain neonatal resuscitation knowledge and skills. However, the current approach to neonatal resuscitation simulation training is time and resource-intensive, and often inaccessible. Digital neonatal resuscitation simulation may present a convenient alternative for more frequent training.Method: Fifty neonatal HCPs participated in the study . This study was conducted at a tertiary perinatal center in Edmonton, Canada from April–August 2019, with 2-month (June–October 2019) and 5-month (September 2019–January 2020) follow-up. Neonatal HCPs were recruited by volunteer sampling to complete a demographic survey, pre-test (baseline knowledge), two digital simulation scenarios (intervention), and post-test (knowledge acquisition). Two months later, participants repeated the post-test (knowledge retention). Five months after the initial intervention, participants completed a post-test using a table-top simulation (knowledge transfer). Longitudinal analyses were used to compare participants' performance over time.Results: Overall the proportion of correct performance increased: 21/50 (42%) passed the pre-test, 39/50 (78%) the post-test, 30/43 (70%) the 2-month post-test, and 32/40 (80%) the 5-month post-test. GLMM and GEE analyses revealed that performance on all post-tests was significantly better than the performance on the pre-test. Therefore, training with the RETAIN digital simulation effectively improves, maintains, and transfers HCPs' neonatal resuscitation knowledge.Conclusions: Digital simulation improved, maintained, and helped transfer HCPs' neonatal resuscitation knowledge over time. Digital simulation presents a promising approach for frequent neonatal resuscitation training, particularly for distance-learning applications. Each year, ~10% of infants worldwide need help to breathe at birth. To safely care for these newborn patients, health-care professionals (HCPs) must master their knowledge and decision-making skills outlined by the neonatal resuscitation algorithm. However, one million infants still die each year from asphyxia at birth . To imprHowever, despite widespread uptake of the simulation-based Neonatal Resuscitation Program (NRP), HCPs remain dangerously underprepared for clinical neonatal resuscitation events due to decay in clinical skills after training . One reaAlternative approaches to traditional in-person simulation, such as digital simulation, may provide a solution. Digital simulation offers a scalable online platform for learners to readily access educational content and achieve many of the learning objectives of traditional simulation-based education, particularly for training HCPs' knowledge, including the recall of the correct sequence of steps for a clinical procedure . DigitalThis study aimed to examine if training with a digital simulation improves HCPs' neonatal resuscitation knowledge over time. This study also aimed to measure HCPs' knowledge transfer from the digital simulation. We hypothesized that HCPs would experience short- and long-term knowledge improvement of the correct steps of neonatal resuscitation after training with the digital simulation and transfer this knowledge into a novel learning environment.Neonatal HCPs were recruited by voluntary sample from the Royal Alexandra Hospital Neonatal Intensive Care Unit (NICU), Edmonton, Canada, a tertiary perinatal center delivering over 6,500 infants annually. Recruited participants included registered nurses, respiratory therapists, doctors, and neonatal nurse practitioners. The demographic characteristics  of the study sample was representative of the HCP population within the NICU. The study was performed at the bedside in the NICU and was approved by the Human Research Ethics Board at the University of Alberta, Edmonton, Canada (Pro00081221). Written informed consent was obtained from HCPs prior to participation.https://www.retainlabsmedical.com/index.html) is a digital simulator for HCPs to practice their knowledge and decision-making of neonatal resuscitation  who completed NRP-recertification within the last 24-months. The study consisted of four different assessments administered over three different timepoints: an initial session (including a pre-test and a post-test), a 2-month follow-up session, and a 5-month follow-up session . Depending on the steps taken by the participant, the infant's health improved, worsened, or remained unchanged . If all steps of the NRP algorithm were performed correctly, the infant's health would improve, the infant would stabilize, and the scenario would end. If participants completed any step of the NRP algorithm incorrectly, the infant would fail to stabilize, and participants would exit the scenario.Participants completed all scenarios individually without assistance. Feedback was only provided through visual and cardiorespiratory assessment of the infant throughout the scenario . However, the 5-month post-test to measure knowledge transfer included two additional actions (obtaining vascular access and administering one dose of epinephrine).The sessment . For eacsessment . A failisessment . Becausepre-test, post-test, 2-month post-test, and 5-month post-test were assessment scenarios. The RETAIN table-top simulation was previously reported as a method for summative assessment of neonatal resuscitation providers , nor had any of the participants previously used the digital nor tabletop simulator described in the present study. The only training scenarios for participants to practice their neonatal resuscitation knowledge occurred during the initial session . In contrast, the roviders .Statistical analyses were performed using RStudio AGPL v3 Desktop Open Source Edition  with R version 4.0.1 . We condpre-test (baseline assessment), training scenarios, and post-test (immediate knowledge improvement). Forty-three participants  completed the 2-month follow-up session, consisting of the 2-month post-test (long-term knowledge retention). Forty participants  completed the 5-month follow-up session, consisting of the 5-month post-test (knowledge transfer).The number of months since participants completed their last NRP course was median (IQR) 9 (5–12) months, and number of years of clinical neonatal experience was median (IQR) 9 (6.4–15.2) years. All participants completed the initial session, consisting of the pre-test (42%), 39 of 50 participants passed the post-test (78%), 30 of 43 participants passed the 2-month post-test (70%), and 32 of 40 passed the 5-month post-test (80%). The distributions of the performance at each timepoint were explored using violin plots from the ggplot2 package , mean performance after 5-months was double the mean performance on the pre-test was significantly associated with the 5-month post-test . Also, the association between the pre-test and the immediate post-test did not reach significance . Results were identical for the Spearman correlation as well.Pearson correlation analyses revealed that the glmer function of the lme4 package in R .GLMM models with dichotomous longitudinal outcomes were fit using the age in R  by maximggplot function from the ggplot2 R package was used to plot the results of the prediction expressed in probabilities shown in pre-test, captured by the differences in starting points displayed on the left side of post-tests. The variability in the intercepts modeled with the random-intercept term indicates that some groups of participants had high model implied probabilities of performing correctly throughout all testing stages, whereas others had lower probabilities throughout all of the four testing points.The geeglm function of the geepack package in R, with binomial distribution and logit link function, was used to fit the GEE models that estimate the population aggregate (average) trend directly to make inferences about the population rather than about an individual. The QIC function of the MuMIn package in R was used to compute the quasi-information criterion (QIC) statistic that guides model selection . The GEE model selection is detailed in The Lifetime risk of mortality is the highest on the day of birth . Birth aSimulation-based education is recommended to address these deficiencies by replicating a real-world clinical situation, while simultaneously maintaining patient safety . SimulatIn contrast, digital simulation presents a promising alternative to overcome traditional time and resource limitations of neonatal resuscitation simulation. Digital simulation may offer a convenient solution for neonatal resuscitation providers to access more frequent training to foster sustained learning . Severalpre-test, likely due to the variety and size of the sample. Therefore, a single session with the RETAIN digital simulator immediately improved performance demonstrated by highly experienced neonatal resuscitation providers. Furthermore, HCPs' improvements were sustained 2-months after training. The proportion of correct performance tended to increase over time, with only small variations. The median of the pre-test was smaller than the mean, suggesting that there are a few participants who performed very well on the pre-test and pulled the mean of the group above the median. Performance on the pre-test tended to be slightly poor, but the trend was reversed for all three post-tests, more so for the immediate post-test and for the 5-month post-test. All post-tests had similar variation, higher than that of the pre-test. Therefore, training with the RETAIN digital simulation resulted in HCPs' long-term knowledge retention.Our participants were experienced neonatal HCPs recruited from a tertiary perinatal care center . When compared to their baseline scores, we observed that HCPs significantly improved their performance after training with the RETAIN digital simulator, although there was some variation in the pre-test and the 2-month post-test. However, the strongest correlation found was between the first (pre-test) and the last (5-month post-test) timepoints. This result may be explained by the different nature of the 5-month post-test; however, this result is promising, considering the 5-month post-test administered a more complex scenario compared to previous tests.We expected the correlation between performance scores at different timepoints to decline. That was the case between the To measure knowledge transfer, we used a table-top simulator previously reported as a valid summative assessment tool to evaluate neonatal resuscitation performance . KnowledThe RETAIN digital simulator provides neonatal resuscitation scenarios of varying difficulty and patient risk factors, accessible by HCPs anywhere and anytime , 11. DigIn contrast, digital simulation offers a feasible approach toward active remote learning, which may be readily taken up by neonatal resuscitation providers during COVID-19. Widespread implementation of digital simulation can provide a practical tool for health-care institutions to transition to effective and clinically relevant remote learning strategies. In consideration of current public health challenges, continuation of health-care education is essential to improve HCP performance so they may continue to provide safe and effective health care for their patients during these unprecedented times.pre-test scores of HCPs who dropped out compared to those who remained. Additional studies are needed to compare digital simulation to other remote learning methods and to measure changes in HCPs' performance in the delivery room after training with digital simulation.The RETAIN digital simulator was not designed with the goal of improving psychomotor practical skills. Therefore, the current version of the RETAIN digital simulator should be used to reinforce rather than replace traditional simulation-based education. It is not clear whether improved performance on a digital simulator indicates competence in providing neonatal resuscitation care. Other limitations of the study include convenience sampling, lack of random assignment and control group. However, our attrition rate was reasonable, considering participants were HCPs on a busy intensive-care unit, which made retesting difficult. Further, there was no difference between After using the RETAIN digital simulator, HCPs demonstrated improved adherence to the neonatal resuscitation algorithm, which was sustained 2- and 5-months after the initial training. HCPs also successfully demonstrated knowledge transfer to a new learning environment. The RETAIN digital simulator may be useful to supplement formal neonatal resuscitation education, with potential application for remote learning during the COVID-19 pandemic.The original contributions presented in the study are included in the article/The study was performed at the bedside in the NICU and was approved by the Human Research Ethics Board at the University of Alberta, Edmonton, Canada (Pro00081221). Written informed consent was obtained from HCPs prior to participation. The patients/participants provided their written informed consent to participate in this study.GS attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. SG, MC, and GS conceptualized and designed the study, designed the data collection instruments, collected data, carried out the analyses, and interpretation, drafted the initial manuscript, and critically reviewed and revised the manuscript. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.https://www.retainlabsmedical.com/index.html). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.GS has registered the RETAIN table-top simulator (Tech ID 2017083) and the RETAIN digital simulator under Canadian copyright (Tech ID – 2017086) and is an owner of RETAIN Labs Medical Inc., Edmonton, Canada (


Neuro-arthropathy is a destructive osteo-articular condition with typical radiological features that can be the result of every disease affecting the sensitive nervous pathways. A 45-year-old man came to the emergency service exhibiting non-traumatic swelling and moderate pain in the left knee. The joint was red and hot at physical examination, and the neurologic tests were normal. He had a medical history of schizophrenia (treated with Olanzapine and Quietapine), diabetes , alcoholism (no B12 deficiency), and obesity (Body Mass Index = 47). Biology was normal except for moderate systemic inflammation (C-Reactive protein 68.1 mg/L [<5 mg/L]). Left knee radiographs showed impacted medial femoro-tibial articular surfaces with “mirror deformity”, sub-chondral sclerosis, bone fragmentation with intra-/peri-articular calcified debris Figure  and joinNeuro-arthropathy is an osteo-articular disorder of uncertain pathogenesis. It is believed that a neuropathic background can result in a major joint destruction due to the loss of pain sensation. The initial injury (micro/macro-trauma), going unnoticed, leads to inflammatory response, unnoticed too, which results in bone surface alteration that favors new injuries. This vicious circle ends in severe joint destruction, with or without bone reformation (hypertrophic or atrophic forms). At the early stage of the disease, the joint presents swelling, redness, and heat, but the radiographic abnormalities are very subtle  and the diagnosis is often missed. Magnetic resonance imaging (MRI) can show medullar edema and is useful to rule out septic complication, especially in the foot (Charcot). A period of rest and avoiding weight bearing can stop the progression to the chronic stage. At this point, the radiographic abnormalities are evident, and can be summarized by the 6 Ds mnemonic: density change (osteopenia/sclerosis), destruction (fragmentation), debris (loose bodies), distension (effusion), disorganization, and dislocation. The treatment can be conservative or surgical. The far most common cause in our modern society is long-standing and uncontrolled diabetes, especially in patients with peripheral neuropathy. By extension, every disease that affects the peripheral or central nervous sensitive pathways can lead to neuro-arthropathy; that includes alcoholism (B12 deficiency), syrinx medullary trauma, and the like.In the absence of any of these more typical causes, the hypothesis of schizophrenia was retained as the cause of this knee neuro-arthropathy. Indeed, this condition is known to affect the pain sensation, with a higher pain threshold. Some schizophrenic patients may suffer from congenital insensitivity to pain .


The regular use of gamma-hydroxybutyrate acid (GHB) can induce GHB-induced comas. Other substance use disorders are associated with alterations in brain structure and impulsivity. Here we aim to investigate if these are also modulated by either regular GHB use or GHB-induced comas.In a sample of human males, structural and diffusion neuroimaging data were collected for 27 GHB users with ≥4 GHB-induced comas (GHB-Coma), 27 GHB users without GHB-induced comas (GHB-NoComa), and 27 polydrug users who never used GHB (No-GHB). The structural brain parameters were analyzed macroscopically using voxel-based morphometry and microscopically using tract-based spatial statistics (TBSS) and tractography. Impulsivity was assessed with the Barrat Impulsivity Scale.i.e., whole-brain TBSS analysis). No macrostructural differences nor microstructural differences, as assessed with tractography, were observed. The GHB-Coma group also reported higher impulsivity, which was more strongly associated with white matter volume and fractional anisotropy in tracts involved in impulse control . GHB use per se was associated neither with differences in brain structure nor with impulsivity.In comparison to the other two groups, the GHB-Coma group showed a higher fractional anisotropy in the body of the corpus callosum and a lower mean diffusivity in the forceps minor (The results suggest that multiple GHB-induced comas, but not GHB use per se, are associated with microstructural alterations in white matter and with higher self-reported impulsivity, which in turn was associated with white matter tracts involved in impulse control. Since it was first synthesized in the 1960s, gamma-hydroxybutyrate acid (GHB) has been regularly used for different therapeutic purposes . Over the.g., the hippocampus), and their functional connectivity with the temporal–parietal lobe regions involved in perception and attention , and the parietal-occipital cortex have been observed    deficiency, a condition known to induce an abnormal accumulation of GABA and GHB in the brain, gray matter atrophy and white matter myelin alterations in the PFC, insula, limbic system  –23. Moresum, CC) , 22–29. sum, CC) , 30.This study aimed to investigate the effects of recreational GHB use and multiple GHB-induced comas on impulse control and brain structure. Self-reported impulsivity was measured with the Barratt Impulsivity Scale (BIS). The macrostructural differences were assessed with voxel-based morphometry analysis (VBM) of structural magnetic resonance imaging (sMRI) data, and diffusion-weighted imaging (DWI) data were investigated with a univariate whole-brain level tract-based spatial statistical analysis (TBSS) or with a region-of-interest (ROI) probabilistic tractography analysis (TrackVis), focused on the ILF, IFOF, and UF, for the assessment of microstructural differences. To distinguish between the effects of recreational GHB use as such and multiple GHB-induced comas, three different groups of participants were recruited: (1) GHB users who had ≥4 GHB-induced comas, (2) GHB users who never had a GHB-induced coma, and (3) polydrug users who never used GHB. We tested the following hypotheses:Regular recreational users of GHB who had multiple GHB-induced comas show higher impulsivity and macrostructural and microstructural brain alterations when compared to GHB users who never had a GHB-induced coma and to polydrug users who never used GHB.Regular recreational users of GHB who never had a GHB-induced coma show higher impulsivity and macrostructural and microstructural brain alterations when compared to polydrug users who never used GHB.n = 81) were recruited for this cross-sectional study through addiction centers in The Netherlands, flyers, internet advertisements, and snowball sampling. Three different groups of male participants, matched on age and education level, were included: 27 GHB users with ≥4 GHB-induced comas (GHB-Coma), 27 GHB users without GHB-induced comas (GHB-NoComa), and 27 polydrug users who never used GHB (No-GHB). The criteria considered in this study were a result of self-reported parameters and urine tests. The inclusion criterion for the GHB-Coma group was >4 GHB-induced comas (to increase the contrast with GHB-NoComa group). The inclusion criterion for the GHB groups was the use of GHB ≥25 times within the 2 years preceding this assessment. The overall inclusion criteria were age (between 18 and 40 years), native Dutch speaker, and male gender  , any coma unrelated to GHB use, and currently under treatment for narcolepsy with cataplexy (since the treatment may involve the use of medication based on GHB). The participants excluded due to low-quality scans were two GHB-Coma and two no GHB cases, respectively. The study procedures were explained prior to the assessments and written consent was obtained from the participants. This study was performed in accordance with the Helsinki Declaration principles  and the Medical Research Involving Human Subjects Act  and approved by the Medical Ethics Review Committee of the Academic Medical Centre   . The pol014_172) .i.e., substance use habits, negative affect, and impulsivity), and structural and functional imaging scans collected as follows: sMRI, resting state (fMRI), episodic memory (fMRI), DWI, working-memory (fMRI), and emotion identification (fMRI). Outside the scanner, the participants performed digitized neuropsychological tests concerning verbal memory, spatial memory, intra-/extra-dimensional set shifting, and probabilistic reversal learning. In this report, we will only present data on impulsivity and brain structure. Other findings have been presented elsewhere . Normally distributed data were evaluated through ANOVA. If not normally distributed, the data were transformed in order to obtain a normal distribution or were evaluated with non-parametric tests  were acquired with a magnetization-prepared rapid gradient echo sequence for spatial normalization purposes. Diffusion-weighted images were acquired in 32 isotropic directions in order to test white matter abnormalities. Each image consisted of 48 transverse slices .Diffusion-weighted and structural images were collected with a 3.0-T Ingenia scanner with a 32-channel head coil . T1-weighted structural images  as implemented on Statistical Parametric Mapping software . Prior to preprocessing, the origin was manually set to the anterior commissure. T1-weighted images were segmented into gray matter, white matter, and cerebral spinal fluid after bias-field correction to remove non-uniformities in intensity, normalized into Montreal Neurologic Institute (MNI152) space, and smoothed using a Gaussian kernel of 8 mm at full-width half-maximum. In addition, total intracranial volume (TIV) was estimated to correct for differences in total brain size.Preprocessing of structural data was conducted using the Computational Anatomy Toolbox 12 . The number of variables representing co-exposure to other drugs in which group differences were observed was adapted to the sample size considered for each neuroimaging method of analysis. TIV was used to correct for differences in brain volume across subjects .www.fmrib.ox.ac.uk/fsl) (a priori defined ROI (tracts) with streamline tractography analysis.Preprocessing of DTI data was conducted with the FMRIB Software Library 5.0.10  . Pre-pro.uk/fsl) –25, 38. Whole-brain white matter differences were assessed with TBSS (FSL) as follows: (1) non-linear alignment of individual pre-processed FA images to a common FMRIB58 FA brain template , (2) averaging of aligned images into a mean FA map of all individual FA images, (3) creation of a mean FA skeleton map and a mean FA mask by computing a white matter tract skeleton into the mean FA map, and (4) creation of an all-skeletonized FA map  thresholding the mean FA skeleton map at 0.2 . All FA www.trackvis.org/download/). For compatibility reasons, data were transformed from nifti to dtk format, and the tensor orientation was flipped around the z-axis . 3D virtual dissections were performed per participant, per tract and per hemisphere, by manually delineating two ROIs per tract on the FA maps of each participant based on established references of regions crossed by specific tract bundles (pFWE < 0.05) for multiple comparisons using a height threshold of p < 0.001 and a Bonferroni correction to account for the multiple ROIs tested.Tractography was performed on the ILF, the IFOF, and the UF based on their involvement in impulse control and proximity to regions where functional alterations were associated with recreational GHB use , 24, 26. bundles , 41. An pFWE < 0.05)  . Two ortp = 0.008). On average, the GHB-Coma group used GHB in higher daily doses , more frequently in the preceding month , and daily during more months  when compared to the GHB-NoComa group. The co-use of recreational drugs was also significantly different between groups , only self-control  remained statistically lower in the GHB-Coma group compared to the GHB-NoComa group and the No-GHB group .There were no significant group differences in age (mean ± SD = 26.51 ± 6.90) and education level  data by using a group-by-impulsivity interaction controlled for IQ, cocaine, other stimulants, ecstasy, sedatives, and TIV  of the GHB-Coma group. In contradiction of our first hypothesis, increased FA and decreased MD are general indicators of white matter integrity. This suggests that the anatomical alterations observed might have been present already before the occurrence of GHB-induced comas and represent a risk factor for the onset and development of heavy chronic use of GHB , 42, 43.In contrast to these TBSS findings, no alterations in white matter integrity were found with tractography. However, TBSS assesses local integrity, whereas tractography assesses mean integrity along the entire tract, suggesting that in the GHB-Coma group, microstructural differences in white matter only occur at a more local level , 51, 52.Lastly, since impulsivity is a common comorbidity of substance use disorders  and a lasting effect of transient unconsciousness, we decided to compare the level of impulse control between the groups , 29, 53.Moreover, it is important to consider the fact that the pre-frontal and limbic parts of the white matter tracts, where alterations were found in this study, are regions rich in GHB binding sites that have been shown to be highly sensitive to neurotoxicity induced by chronic GHB intake  , 29, 53.a priori trait that biased the motivational system of this group towards the immediate reward provided by GHB use. However, all groups were matched for education level and, therefore, the lower IQ in the GHB-Coma group is most likely a result from the repeated GHB-induced comas. Notwithstanding, IQ was used as a covariate in the analysis. Moreover, the last few years have witnessed an increase in the use of GBL . However, the participants included in this study reported solely the use of GHB in its absolute form  and pharmacotherapy   , 60. FinAll datasets generated for this study are included in the article.The studies involving human participants were reviewed and approved by the Medical Ethics Review Committee of the Academic Medical Centre. The patients/participants provided their written informed consent to participate in this study.MM, WV, and GV designed the study. MM, NP, and YD collected the behavioral and neuroimaging data. AS contributed in the collection of the data. FR performed the neuroimaging and statistical analyses. FR and GV were responsible for the interpretation of the data. FR wrote the manuscript. FR, MM, WV, GV, YD, and AS have revised and approved the submission of the final manuscript.This research was funded and supported by the Ministry of Health, Welfare, and Sport of The Netherlands.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose: The purpose of the current study was to evaluate the ability of magnetic resonance (MR) radiomics-based machine-learning algorithms in differentiating glioblastoma (GBM) from primary central nervous system lymphoma (PCNSL).Method: One-hundred and thirty-eight patients were enrolled in this study. Radiomics features were extracted from contrast-enhanced MR images, and the machine-learning models were established using five selection methods , eXtreme gradient boosting (Xgboost), and Gradient Boosting Decision Tree) and three radiomics-based machine-learning classifiers . Sensitivity, specificity, accuracy, and areas under curves (AUC) of models were calculated, with which the performances of classifiers were evaluated and compared with each other.Result: Brilliant discriminative performance would be observed among all classifiers when combined with the suitable selection method. For LDA-based models, the optimal one was Distance Correlation + LDA with AUC of 0.978. For SVM-based models, Distance Correlation + SVM was the one with highest AUC of 0.959, while for LR-based models, the highest AUC was 0.966 established with LASSO + LR.Conclusion: Radiomics-based machine-learning algorithms potentially have promising performances in differentiating GBM from PCNSL. Glioblastoma (GBM) and primary central nervous system lymphoma (PCNSL) are considered as the common primary brain tumors, which share similar radiological characteristics but diverse in therapeutic strategies –3. The sTexture analysis (TA) refers to a number of a set of mathematical methods describing the features of images, with which non-visual information could be represented with analyzable pixel intensities and the spatial distributions , 9. It hThe purpose of the present study is to discriminate GBM from PCNSL with radiomics-based machine-learning algorithms in contrast-enhanced T1-weighted (T1C) imaging. In addition, we evaluated different combinations of selection methods and classifiers, trying to make comparison of models' performances.The patients were selected from neurosurgery department by reviewing the electronic medical records between 2015 and 2018. The including criteria of patients were as follows: (1) pathologically confirmed on GBM or PCNSL; (2) undertook MR scan before any tumor biopsy or surgery; (3) newly diagnosed GBM or PCNSL. Some patients were excluded because of the history of intracranial surgery or irrelevant intracranial diseases. In total, 138 patients  were enrolled from the institution database, including 76 patients diagnosed with GBM and 62 diagnosed with PCNSL.The MR images were collected from the PACS system in the radiological department. We focused on conventional MR sequences, including T1-weighted image (T1WI), contrast-enhanced T1-weighted (T1C) imaging, T2-weighted image (T2WI), and fluid-attenuated inversion recovery, considering that the advanced MR sequences were not commonly used in our institution. After the initial evaluation of images, T1C was selected as the study sequences with rather clear description of the boundary between the tumor tissues and normal brain tissue .2, time echo = 30 ms, 30 axial slices, slice thickness = 5 mm (no slice gap), flip angle = 90°, and 200 volumes in each run. Gadopentetate dimeglumine (0.1 mmol/kg) were taken as the contrast agent. The multi-directional data of contrast-enhanced MRI were collected with the continuous interval time of 90–250 s.The preoperative MR scan was conducted with 3-T GE MRI system with an eight-channel phase-array head coil. The protocols of the contrast-enhanced T1-weighted imaging were time repetition = 2,000 ms, field of view = 240 × 240 mmAll procedures involving human participants were in accordance with the ethical standards of the institutional and/or national research committee. The Ethics Committee of Sichuan University approved this retrospective study. Written informed consent was necessary before radiological examination (written informed consent for patients <16 years old was signed by parents or guardians) for all patients. They agreed to undertake the examination if needed and were informed that the statistics (including MR image) might be used for academic purposes in the future.http://www.lifexsoft.org) under the supervisions of senior radiologists. By manually drawing along the tumor tissue slice by slice, the software automatically retrieved 3D-based texture features from two sets of orders with default settings , least absolute shrinkage and selection operator (LASSO), eXtreme gradient boosting (Xgboost), and Gradient Boosting Decision Tree (GBDT).The purpose of machine learning was to establish and train the models to discriminate GBM from PCNSL with radiomics features extracted from T1C imaging. Three classifiers were tested, including linear discriminant analysis (LDA), support vector machine (SVM), and logistic regression (LR). Thus, 15 diagnostic models were evaluated with different combinations of selection methods and classifiers. The models were trained with the statistics of the training group and tested in the validation group. Sensitivity, specificity, area under the receiver operating characteristic curve (AUC), and accuracy of each model were recorded for evaluation. On application of each model, the cycle of training-validation was performed 100 times to obtain the realistic distribution of classification accuracies. The flow chart of the study is represented in https://scikit-learn.org/stable/).The models were programmed using Python Programming Language in this study. The models were directly established with default hyperparameter settings of scikit-learn packages  and frequent in GBM (44.4%) . AdvanceComparing with GBM, permeable neovascularization and higher degree of cellularity were more likely to be observed in PCNSL, which theoretically provide the mechanism of TA-based image discrimination –24. In oThe possible explanation for the improvement was the performance improvement in selection method. Radiomics analysis involved large amounts of features, but machine learning required the most suitable parameters. Previous researchers selected parameters with F-statistic approach into SVM classifier, while we selected with distance correlation, RF, LASSO, Xgboost, or GBDT approach. The combination of LASSO + SVM represented similar discriminative performance such as in the previous study with AUC of 0.822. Besides performances, we can also find that the selection methods were also important to the model stability. Over-fitting is a problem that should be avoided in designing the machine-learning models, which happens when the models catch inaccurate values in the data and the noisy data. Our results suggested that over-fitting probably occurred when using RF, Xgboost, and GBDT as selection methods. Perhaps the features selected with these methods contained too much noise and led to the over-fitting of models.As for the classifier selection, the purpose of enrollment of three classifiers was to choose the suitable one in discriminating GBM from PCNSL. The results suggested that with suitable features, all of them could represent discriminative ability. It is worthy to note that although we chose Distance Correlation + LDA as the optimal model, some models (like LASSO + LDA and LASSO + LR) also represented pretty similar discriminative performances. The model Distance Correlation + LDA was chosen as the optimal one because it has the minimal difference between sensitivity and specificity compared with LASSO + LDA and LASSO + LR. However, given that all classifier/feature selection methods investigated seem to perform quite comparably and variance in AUC may be partially attributed to small statistical group, the additional gain in information by comparing machine-learning models was quite limited and carefully interpreted. Future investigations with larger sample sizes are required to address this problem and verify our results.There were several limitations to our study. First, the isolated evaluation of T1C image is not representative of the real clinical work given other sequences  could also be useful. Second, the diagnostic performance of radiomics-based machine learning was not compared with other advanced MRI technology. Third, the study cohort is not large enough, requiring study with a large population to verify our results. Forth, the machine-learning classifier was not validated in the other dataset. Considering the considerable variability in images acquired with various MR scanner at different institutions, we cannot guarantee the diagnostic ability of our machine-learning classifier for external datasets. However, the image processing and analysis protocol were open-source packages, meaning they should be validated and reproduced with other datasets.Radiomics with machine-learning algorithm technology represented promising ability in differentiating GBM from PCNSL.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of Sichuan University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.XM participated in conceptualization and revised some intellectual content in the manuscript. CC collected MR image, participated in MRI features extraction, and drafted this manuscript. XO collected MR image and participated in MRI features extraction. JW deployed the machine-learning algorism and responsible for statistical analysis. AZ participated in the most revision work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cells are minimal functional units in biological phenomena, and therefore single-cell analysis is needed to understand the molecular behavior leading to cellular function in organisms. In addition, omics analysis technology can be used to identify essential molecular mechanisms in an unbiased manner. Recently, single-cell genomics has unveiled hidden molecular systems leading to disease pathogenesis in patients. In this review, I summarize the recent advances in single-cell genomics for the understanding of disease pathogenesis and discuss future perspectives. The heart constantly responds to hemodynamic overload. Cardiomyocytes, which are the principal components of the pump function of the heart, are required to maintain cardiac homeostasis by adapting appropriately to this stress. However, sustained exposure to pathological stress disrupts the adaptive mechanisms of cardiomyocytes, leading to heart failure. Understanding how each cardiomyocyte responds to various stimuli at the single-cell level will help to elucidate the pathogenesis of heart failure.Cardiomyocytes are cylindrically shaped, around 120-μm long and 30-μm wide. Because of this large size, See et al. decided to isolate nuclei from cardiomyocytes using a microfluidics system and conducted single-nucleus RNA-sequencing (RNA-seq) to reveal the activation of cell-cycle regulators and novel long noncoding RNAs in diseased cardiomyocytes .We applied this system to a mouse model of pressure overload-induced heart failure and obtained single-cell transcriptomes of cardiomyocytes isolated during the progression of heart failure . WeighteTo recover the spatial information lost in single-cell RNA-seq analysis, we established a single-molecule RNA in situ hybridization assay, which enables quantification of each mRNA at the single-cell level , and revWe and others have applied single-cell genomics and used cell-type classification, trajectory inference, marker identification, spatial analysis, and clinical assessment to further our understanding of disease pathogenesis. However, conventional single-cell RNA-seq analysis is not sufficient for obtaining the information necessary for a deeper understanding of molecular behavior. Recently, several studies have opened new avenues by focusing on cell–cell communications, spatial single-cell omics, barcode lineage tracing, single-cell multi-omics, multiple perturbations with single-cell readout, immunoprofiling, and clinical application Fig. . In thisSeveral types of cells interact with each other via a variety of signaling molecules to generate organ-level functions. By integrating the single-cell expression profiles of ligands and receptors in developing lung tissue with a ligand and receptor interaction database, Cohen et al. generated a cell–cell communication map and identified eosinophils as essential signal mediators in the lung . EosinopGiladi et al. reported an approach for sequencing physically interacting cells (PIC-seq), which integrates cell sorting of physically interacting cells (PICs) with single-cell RNA-seq data, to comprehensively investigate the functional nature of PICs and identify the signaling molecules associated with these interactions . In the Cell–cell communications also occur at the level of the organism. Ma et al. obtained ~210,000 single-cell transcriptomes from several organs  from young and aged rats with or without calorie restriction and analyzed the changes in cellular distribution and cell-type-specific expression profiles . They reCell–cell communications can be assessed more accurately by in situ analysis with preservation of spatial information in tissues. Individual mRNA molecules can be detected accurately in cells with single-molecule fluorescence in situ hybridization (FISH) . Eng et Multiplexed proteome approaches also enable quantitative analysis of protein expression levels in situ at the single-cell level . GoltsevFos and ribosomal RNAs are activated at the injured position; at 3 days after injury, cell-cycle-related genes are activated in microglia and macrophages in remote regions; at 2 weeks after injury, these cells are replaced to fill the injured position. These cells also showed the activation of genes involved in the development of oligodendrocytes.Rodriques et al. developed Slide-seq, a method for transferring tissue sections onto a surface covered with DNA-barcoded beads in known positions . Using tISL1 and STMN2, and Schwann cell precursors, marked by ALDH1A1. Both cells emerge in the outflow tract, but only the latter localizes in the subepicardial interstitium during heart development. Even in the outflow tract, the former emerges only in the early phase, whereas the latter is found only in the late phase.Lundeberg et al. developed spatial transcriptomics, an approach similar to Slide-seq , and perRecently, a variety of tissue clearing methods have been developed that can be applied to single-cell protein expression analysis in several organs . AlthougCaenorhabditis elegans and identified 502 cell types and 1068 developmental trajectories [Trapnell et al. developed Monocle, an algorithm-based application for pseudotime analysis and inference of the linear or bifurcating trajectories of an individual cell’s progress through differentiation , 31; to ectories . They fuectories .Additional information, such as the ratio of reads mapped to introns and exons, RNA metabolism, and protein expression profiles, enables trajectories to be inferred more accurately. On the basis of the concept that transcriptionally active cells have more unspliced mRNAs, La Manno et al. developed Velocyto, an algorithm for inferring trajectories using the ratio of reads mapped to introns and exons . They apBecause Monocle and Velocyto infer cellular trajectories by using single-cell information derived only from RNA molecules, these algorithms cannot accurately reconstruct trajectories in cell-state transitions such as endothelial–mesenchymal transition. Recently, on the basis of the concept that daughter cells generally have the same genome, lineage tracing analysis using DNA barcode technology, a method of lineage identification that uses a short section of re-writable DNA, has been advancing . ApproacThe simultaneous detection of DNA sequences and RNA expression profiles enables the identification of disease-causing variants and their association with gene expression. There are novel methods to simultaneously extract information from DNA and RNA –51. By pCALR-mutated myeloproliferative neoplasms, identifying an association between the activation of the unfolded protein response and NF-κB pathway with CALR mutations [The detection of single nucleotide variants using abundant single-cell RNA-seq data is an applicable and cost-effective method for identifying expressed variants, inferring sub-clones, and deciphering genotype-phenotype relationships . Enge etutations .Drosophila embryos identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and Polycomb-independent borders. Deletion of the Polycomb-independent borders leads to ectopic contacts between enhancers and promoters, resulting in aberrant gene expression and developmental defects. Katzenelenbogen et al. developed intracellular staining and sequencing  that enables simultaneous detection of the intracellular signaling and protein state as well as the cellular transcriptional profiles, and identified Arg1+ Trem2+ regulatory myeloid cells, which control tumor growth [Given that gene expression is regulated by the epigenome, simultaneous analysis of the transcriptome and epigenome leads to a deeper understanding of gene regulation. There are methods to simultaneously detect combinations of RNA expression and DNA methylation , chromatr growth .CBL and CNN1 for driving erythroid differentiation [HSPA5 and GATA1 by ~50% is sufficient to induce a near maximal transcriptional response and growth defect, whereas a larger reduction of other genes is required for a similar effect, suggesting the sharp transition in cellular behavior at gene-specific expression thresholds [A combination of CRISPR/Cas9-based genetic screening and single-cell omics analysis enables comprehensive and detailed functional analyses –70. Normntiation . By titrresholds . Reploglresholds .Multiple perturbations with single-cell readout has been developed to analyze epigenetic regulation , enhance+ T cells [+ T effector memory CD45RA+ cells and disease severity [+ T cells can kill autologous tumors in an MHC class II-dependent manner and are suppressed by regulatory T cells. A gene signature of cytotoxic CD4+ T cells predicted the clinical response of patients with metastatic bladder cancer treated with anti-PD-L1.The diversity of the vertebrate adaptive immune system is based on somatic rearrangements of V(D)J genes encoding the T-cell receptor (TCR) α and β chains; therefore, simultaneous analysis of TCR sequence  and gene expression from individual cells provides a deeper understanding of molecular behavior in the adaptive immune system. By integrating single-cell transcriptomes with clonal information during the development of the human thymus, Park et al. identified a strong bias in V(D)J usage shaped by recombination and multiple rounds of selection, including a TCRα V-J bias for CD8 T cells . Through T cells . Gate etseverity . The macseverity  and clonseverity . These CStoeckius et al. developed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), a method in which oligonucleotide-labeled antibodies are used to measure the expression levels of surface proteins, which is essential for immunoprofiling . Granja + cells in which DNA from human herpesvirus 6b is detected [+ cells in which FOS-Jun, PU.1, and CEBP are activated to regulate immune-related gene expression [+ IL11+ inflammatory fibroblasts as being associated with resistance to anti-TNF treatment, and deployed single-cell co-expression analysis to highlight putative causal genes for inflammatory bowel disease [Single-cell genomics has been utilized for delineating the molecular behavior of rare clinical samples and their relationship with patients’ phenotypes . Velmeshdetected . They alpression . Smillie disease . By comb disease . They alFurther utilization of single-cell genomics analysis using clinical samples to dissect pathology is advancing, but the importance of bulk sample analysis, which does not require specialized equipment and rigorous cell isolation and enables the processing of many samples, will be maintained. There are algorithms to characterize cell-type composition across subjects from bulk RNA-seq data using single-cell RNA-seq profiles as references –97. WangThe batch effect, which is caused by differences in the conditions of sample collection and preservation, the efficiency of cDNA library synthesis, or the number of sequencing reads, should be reduced as much as possible to integrate multiple datasets for large-scale single-cell genomics analysis. Data integration algorithms such as LIGER , Seurat + memory T cells [In the near future, the number of studies integrating single-cell genomics with deep phenotyping  or asses T cells . Srivats T cells . They prHistone modifications and alternative splicing are critical for transcriptional regulation; therefore, the development of methods to analyze them quantitatively at single-cell resolution will lead to a deeper understanding of the molecular mechanisms underlying transcriptional regulation in clinical samples. Henikoff and colleagues developed cleavage under targets and release using nuclease, which is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein–DNA complexes into the supernatant for sequencing, and reduced the number of cells required for epigenomic analysis , 109. HaAs I have summarized in this review, single-cell genomics has been combined with a variety of technologies and has uncovered hidden molecular mechanisms in several biological phenomena, including development, physiology, and pathophysiology. In the near future, the integration of multidimensional datasets obtained through single-cell genomics approaches will have a major impact on biological research and clinical pathology. I fully expect that the implementation and expansion of single-cell genomics will lead to vast improvements in the diagnosis, stratification, and treatment of patients worldwide.


SARS-COV-2 has created one of the most massive pandemics in modern history. There is a rapid accumulation of data on its epidemiology, clinical course, diagnosis, management, and complications. One of the sequelae of COVID-19 pneumonia and acute respiratory distress syndrome (ARDS) is pulmonary fibrosis. There is a dearth of accurate data on the prevalence of pulmonary fibrosis post-COVID-19. We report a patient who developed dyspnea secondary to pulmonary fibrosis after successful treatment of COVID-19 pneumonia. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus behind the most massive pandemic of the decade, has resulted in 33 million infections and nearly 1 million deaths globally as of September 2020. The pandemic currently has a variable epidemiologic course, with a rise in the number of new cases in some countries and a decline in others [One of the complications of COVID-19 pneumonia and ARDS is pulmonary fibrosis . AlthougA 60-year-old gentleman, known pre-diabetic, presented with a dry cough for three months. The cough was continuous, subsiding only for short periods during the day with no diurnal pattern. The patient also complained of a progressively increasing dyspnea on minimal exertion. There was no associated fever, weight loss, or night sweats. He was not taking any medication. The patient was a non-smoker and did not consume alcohol.He was treated for moderate severity COVID-19 pneumonia four months before his admission. His presentation at that time was with fever, cough. Chest X-ray revealed discrete airspace consolidation areas in the bilateral lower and mid zones a. The paThe patient remained symptom-free for around one month before developing the dry cough. The cough progressively worsened over the next three months until he sought medical advice. He was afebrile and hemodynamically stable, saturating normal on room air. Physical examination was significant for bilateral basilar crackles with decreased air entry at the lung bases. The patient had desaturation to 90 % on room air when walking for 10–15 meters. A chest X-ray on this admission showed improvement in the opacities previously seen b.Mycoplasma pneumonia, Legionella pneumophila, and Chlamydia pneumonia). Nasopharyngeal polymerase chain reaction (PCR) for common respiratory viruses  were negative. A COVID-19 re-infection was considered as a possibility. However, a SARS-CoV-2 nasopharyngeal reverse transcription Polymerase chain reaction (RT-PCR) via the GeneXpert system came back negative. The patient was clinically euvolemic . Pulmonary tuberculosis was ruled out with negative acid-fast bacilli smears, culture, and PCR (via Xpert MTB nucleic acid amplification).The patient’s basic blood profile and biochemistry were normal . InfectiA high-resolution computerized tomography (HRCT) scan of the chest revealed bilateral interlobular septal thickening, traction bronchiectasis, and honeycombing with scattered reticular and ground-glass infiltrations, mainly at the lower lung fields and bilateral apex . These fThe patient was discharged in a stable condition with a resolving cough. He is planned to be seen in the pulmonary clinic for pulmonary function tests and possibly start antifibrinolytic therapy.The current pandemic of the novel coronavirus SARS‐CoV‐2, causative agent of the COVID-19 infection , emerged from China and is currently affecting almost all countries globally . SARS-CoThe COVID-19 infection mainly presents with respiratory symptoms inducing a flu-like illness with fever, cough, and asthenia. SARS-CoV-2 can cause severe lung injury. In the high‐risk population like the elderly or those with multiple comorbidities, the virus has a higher prevalence of severe interstitial pneumonia, ARDS, and multi-organ failure. Affected individuals display a variable extent of dyspnea and radiological changes ,7. AlthoPatients affected with pulmonary fibrosis commonly complain of dry cough, fatigue, and dyspnea. Weight loss is expected with physical deconditioning. The deconditioning leads to a decreased functional capacity, a decreased quality of life, and may lead to loss of income. Timely and adequate treatment of COVID-19 infection and other coronaviral diseases does not guarantee prevention from pulmonary fibrosis development later on . SARS-CoAlthough mainly it is considered idiopathic, the inflammatory changes in the lungs secondary to the viral infection can lead to fibrotic changes. This can be more likely with ARDS than pneumonia due to the diffuse alveolar damage and resultant type II pneumocyte hyperplasia . PulmonaThe etiology of pulmonary fibrosis is multifactorial and depends on age, smoking, viral infection, drug exposure, and genetic predisposition ,14. MediOne of the hypotheses regarding aggressive disease courses amongst the elderly population is the increased pro-inflammatory cytokines compared to the younger population. The similarity of cytokine profiles in IPF and COVID-19 infection suggests analogous pulmonary fibrosis’s pathophysiology in these diseases . More thWith an already increasing prevalence of pulmonary fibrosis from other causes, post-COVID-19 lung fibrosis poses a considerable morbidity burden considering the number of infections worldwide . ManagemThe current discharge criteria of COVID-19 infection is similar to any other infectious disease. It mainly comprises symptom improvement, laboratory evidence of SARS-CoV-2 viral clearance from the body, and serology (IgG) if available [Post-COVID-19 fibrosis is one of the emerging complications of COVID-19 pneumonia and ARDS. It is estimated to be prevalent in around one-third of COVID-19 infected hospitalized patients. More extensive studies are needed to investigate this occurrence and test the efficacy of already tested drugs (for idiopathic pulmonary fibrosis) such as antifibrotics for post−COVID-19 fibrosis. We suggest doing a 6-minute walk test to assess exertional desaturation in patients who are fulfilling other discharge criteria for COVID-19 pneumonia and ARDS.NoneWritten informed consent was obtained from the patient for publication of this case report and accompanying imagesThis manuscript is original work and has not been submitted or is not under consideration for publication elsewhere. All the authors have reviewed the manuscript and approved it before submission. None of the authors have any conflict of interest from publishing this work.Mousa Ahmad Alhiyari: Conceptualization, Writing - original draft. Fateen Ata: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Mohd Islam Alghizzawi: Writing - review & editing. Ammara Bint I Bilal: Writing - review & editing. Ahmad Salih Abdulhadi: Supervision, Writing - review & editing. Zohaib Yousaf: Methodology, Writing - review & editing, Supervision.This manuscript is original work and has not been submitted or is not under consideration for publication elsewhere. All the authors have reviewed the manuscript and approved it before submission. None of the authors have any conflict of interest from publishing this work.


Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both  Non-viral gene therapy holds great premises as it is assumed to be less toxic for the host and much safer in terms of gene delivery compared to viral vectors .clinicaltrials.gov accessed on 9-October-2020) there were 5,013 (60%) studies reported on “general bone diseases,” 1,034 (11%) on the “hip”-joint, 600 (7%) studies on “rotator cuff,” 1,002 (12%) studies on “tendon” repair, 337 (5%) studies on “intervertebral disk degeneration” (IVD), and 379 (5%) studies on cartilage repair (“cartilage”)  . Howevertilage”) . Finallytilage”) , 2012. Mtilage”) . Thus, nMany of the alternate approaches are less efficient than viral delivery systems  and due in vitro and in vivo non-viral methods being utilized and more specifically in cartilage, the intervertebral disk and bone and gaps/areas that need to be addressed to move these non-viral strategies forward.In orthopedic research there are a number of significant health burdens that urgently warrant better therapeutic solutions. In addition to bone metabolic diseases, this also includes problematic musculoskeletal degenerative pathologies of cartilage, tendons, and ligaments, as well as the intervertebral disks (IVDs) of the spine. It has been identified that osteoarthritis (OA)  and low in vitro propyl]-N,N, N-trimethylammonium chloride (DOTMA). However, due to initial limitations associated with non-specific protein binding  is a physical method based on the application of high voltage pulses for a short duration to facilitate cellular uptake of nucleic acids or drugs. The concept of electroporation was pioneered by  in skin , lung , estimation of the interval between the injection of therapeutics and the delivery of electrical pulses, electrode geometry and tissue properties to increase the efficiency of electroporation  and low voltage (LV) pulses have been studied, and their effects have been tested for electropermeabilization.poration . The mecVarious electrodes are used depending on the target site and are of different size shapes and made of different materials. Most commonly, the electrodes are made of stainless steel, copper, titanium, and they differ in their electrical conductivity, price, and corrosion . For theExtracellular Vesicles (EVs) are cell-derived, lipid membrane enclosed nanoscale particles capable of packaging proteins, lipids, and genetic cargo such as DNA and various RNAs as summarized by in vivo through pathways such as lysosomal degradation, endosomal pathway, phagocytosis, or degradation by macrophages as reviewed in trans-membrane MHC proteins and longer shelf life , integrins, and cell-specific receptors for cell-to-cell communication and internal cargo as described in elf life . DespiteFirstly, there are many underexplored areas in EV research, such as their population heterogeneity, differences in isolation methods, and reproducibility, as described in Current EV related research has primarily focused on MSC derived exosomes, and many are in clinical trials for the treatment and repair of soft tissues . Very reSynthetic polymers, both degradable and non-degradable, have several characteristics that make them suitable for gene vector delivery, including biocompatibility, low immunogenicity, high affinity for nucleic acids, improved stability in biological fluids, and the ability to be engineered to mediate cellular entry and endosomal escape  . Moreovein vivo thanks to their high transfection efficiency and ability to bypass endocytosis to enter the cytosol, especially when compared to synthetic transfection methods such as lipo/polyplex-based carriers , or other anabolic genes such as insulin-like growth factor-1 (IGF-1) (Articular cartilage degeneration is a severe pathology and affects about three out of 10 people worldwide . There ioperties . Most me (IGF-1) . It has  (IGF-1) . Address (IGF-1) .L-lysine) core covalently linked to a receptor-ligand (transferrin) and addition of cationic liposomes  conjugated PEI/DNA particles was investigated in a rabbit knee joint OA-model  has been applied successfully on primary chondrocytes in a high throughput format . Earlierin vitro and in vivo  (in vitro to transfer EVs to MSCs in co-culture (Extracellular vesicles were used successfully to thrive differentiation of MSCs toward chondrocytes  on EVs) . It has  on EVs) . It coul on EVs) . It also on EVs) . In chon on EVs) . Thus, M on EVs) . Chondro-culture . On the -culture . It was -culture .In vivo, particular challenges persist in overcoming the barriers of GAG and other ECM components to reach the chondrocytes with EVs or other non-viral vectors.To summarize, there were many studies conducted in the area of cartilage repair . Unsolved issues concern how EVs interact with components of the ECM of cartilage. It seems clear that hyaluronic acid (HA) and GAGs, such as chondroitin sulfate, are involved in the regulation of EVs and MVs activity . Of greain situ; however, this is still an emerging field with relevant studies discussed below, described in The IVD is the largest avascular and aneural organ in the human body. It is a joint between adjacent vertebrae in the spinal column and facilitates flexion, extension, and rotation of the spine while relying on the diffusion of nutrients through the cartilage endplate of the vertebral body . As a coin vitro focusing on efficiency, safety, and optimal dose. Out of the seventeen agents assessed, they identified “LT1” as the most efficient and least toxic when compared to other lipid-based agents. When culture medium without antibiotics, buffers, and amino acids was used, including hyaluronidase pre- and post-transfection, these changes to the transfection protocol increased efficiency while maintaining viability. Yet, when compared to the adenoviral associated gene delivery controls, LT1 transfection was significantly less efficient than viral delivery, warranting a need for further optimization of these transfection methods.Lipid-based gene delivery systems were amongst the first non-viral methods used to investigate the effects of gene transfection on IVD cells. in vitro. To determine the potential of siRNAs to knockdown gene expression in nucleus pulposus cells isolated from rats and human patients with scoliosis, these cells were co-transfected with reporter luciferase plasmid Firefly and its corresponding siRNA using lipofectamine  to examine effects on cellular lifespan  ectamine . The explifespan . hTERT sin vitro and in vivo. Transfection of liposomal siRNA for Caspase 3 and A Disintegrin and Metalloproteinase with Thrombospondin motifs-5 (ADAMTS5) was first optimized in a human hepatocellular carcinoma cell line in vitro followed by injection of Caspase 3 and ADAMTS5 siRNA alone or in synergy into a rabbit IVD puncture model -2-aminoehtyl]aspartamide) [PEG-b-PAsp(DET)] and poly(N-isopropylacrylamide)-block-PAsp(DET) [PNIPAM-b-PAsp(DET)], which they termed “mixed polyplex micelles” (MPMs). These MPMs demonstrated high resistance to nuclease activity and protein absorption including significantly higher gene transfection efficiency in nucleus pulposus cells when compared with single block polymers [PEG-b-PAsp(DET)] in vitro and in vivo. Furthermore, when MPMs were loaded with heme oxygenase-1 (HO-1), an anti-oxidant and anti-inflammatory, and used to treat nucleus pulposus cells previously stimulated with IL-1β in vitro, decreases in matrix metalloproteinase 3 (MMP3) and cyclo-oxygenase-2 (COX-2) were observed. These effects were reproduced in an IVD degeneration rat tail model where MPMs loaded with HO-1 were more effective an decreasing the inflammatory response and restoring glycosaminoglycans (GAG) when compared to the single block polymer loaded vectors. The authors of this study went on to develop new synthetic polymer-based non-viral gene delivery systems for treating IVD degeneration. One involved nano-sized polyplexes that self-assemble into a double-shell structure, which are then encapsulated in biodegradable nano-spheres and co-injected with nanofibrous spongy microspheres, providing a two-stage delivery system with both temporal control and highly efficient delivery of pDNA  and gene targets with high efficiency and low cytotoxicity. The next steps could include longer-term studies (>12 months) and scaling-up to relevant larger animal models of IVD degeneration such as the sheep, goat, or dog.Synthetic polymer-based gene vectors are attractive alternatives for non-viral gene delivery when compared to viral vectors as they demonstrate low immunogenicity, have tunable structural and surface components, and can be synthesized on a large scale at relatively low-cost . A limit of pDNA . This syin vitro. Studies by Bucher et al. used Nucleofector technology to electroporate human MSCs with growth factor differentiation factor 5 (GDF5) to transplant these cells in a degenerate bovine IVD organ culture model  or by the presence of specific disease states. They were going further from a healthy state of bone tissue results in clinical cases with an increase in morbidity and mortality . In thisin vivo setting, high doses must be injected/administered to address the issues related to the brief half-life of the growth factors  and degradation must be limited. Secondly, to be efficient, the pDNA has to cross both cell and nuclear membranes to enter the nucleus. Thirdly, the pDNA has to be released from any possible transfection complexes  , which ioduction . Recentloduction . Futhermoduction . In the oduction  or more oduction .TM and Lipofactamine 2000TM, have been widely used in research for several years due to their high and stable transfection efficiencies and commercially availability. On one hand, we can notably cite FuGENE6, which was used to transfer the gene TGF-β1, an osteoinductive growth factor into osteoblasts  and demonstrated superior tissue regeneration when compared to empty scaffold and empty calvarial defect groups. Another documented polyplex is the combination of PEI /pBMP-2, in association with a poly(ε-caprolactone) scaffold. This combination was applied to initiate in vitro differentiation of myoblasts  with pDNA . The useyoblasts . With opin situ. The goals of this study were to guide stem cells through osteogenic lineage with localized and sustained RNA release -block-catiomer (PEG-b-P[Asp-(DET)]) and a CaP-cement scaffold. This system has a high bio-compatibility rate with plasmids encoding osteogenic factors, activin receptor-like kinase 6 (caALK6) together with RUNX2 . With th release .in vitro osteogenic differentiation, and in vivo bone formation  and adipo-tissue-derived stromal cells (ASCs), alone, or in combination with other factors  low penetration depth, (II) high levels of cell death and tissue damage, (III) chances of off-target effects, and (IV) risk of particle migration. Doing ex vivo transfections could be one way to overcome those issues. In that case, the DNA is not directly transferred into the body to the cells of interest; however, in a multiple steps protocol, the desired host cells are (I) isolated from the body, (II) transfected in vitro followed by a selection, and (III) and “grafts” back to the host to act as protein factories or directly as bone-forming cells. The two main advantages compared to in vivo transfections are the step pre-selection of the cells of interest and the post-selection of the transfected cells. This step of quality control of the used cells increases the safety of this NVGD strategy. As safety is one of the main concerns in bone regenerative medicine, the ex vivo NVGD model seems to be more potent at the moment in the context of clinical applications 16GRGDSPC chemically linked to a bone scaffold made with PLGA. Applying this TGF-β1 functionalized scaffold to rabbit critical size bone defects significantly increased bone regeneration compared to control groups  . In thisl groups .To combine many of the beneficial effects of NVGD methods, hybrid delivery systems can be an attractive approach, in particular, lipid and polymer integrated materials. PEI modified with linoleic acid and combined with different scaffolds such as collagen and gelatine as vehicles was used to study the expression levels of FGF-2 and BMP-2 after implantation in rat subcutaneous pockets . AnotherAnother option could be to combine natural and synthesized polymers to optimize NVGD systems. A NVGD consisting of a positively charged gene vector within gelatine microspheres and combined with a hydrogel of a crosslinked oligo (PEG-fumarate) (OPF) was used to investigate the effects of pBMP-2 in a critical-size rat cranial defect model on bone formation . Surprisin vivo implantation of gene activated scaffolds/hydrogels/matrices/complexes at sites of bone defect was linked with expression of pDNA and retention for at least 6 weeks. This was followed by the induction of newly formed bone in a reproducible, stable, time-dependent, and dose-dependent manner  has been applied by many studies with relatively high efficiencies, both ic cargo .in vitro with some studies using organ culture or in vivo rat or rabbit models. The type of vectors that have been investigated range from anti-inflammatory/fibrotic agents, siRNA targeting anti-apoptotic/catabolic enzymes, or discogenic growth factors and transcription factors. For the disk, specific considerations that apply include transducing a relatively acellular tissue. These particular tissue regions may require different vectors (NP versus inner or outer AF), ECM (negatively charged proteoglycans), and disease state. Most in vivo studies have focused on utilizing synthetic polymers with some success and therefore highlighting these NVGD methods (For the IVD, NVGD methods have been primarily investigated  methods . HoweverBG initiated the review, performed the major literature search on PubMed, wrote the sections on cartilage repair, drafted major parts of the MS, painted and created the figures, and provided the funding. ST provided the sections on EVs. DP provided the chapters on lipofection and IVD regeneration, language in the “Discussion and Conclusion” section, provided the funding, and edited the manuscript. JG provided the section on bone regeneration. AG provided the text on the non-viral gene therapy methods and electroporation. AC approved and edited the text. NH-C and AS-P provided the sections on the advantages and disadvantages of NVGD and introduction to synthetic polymer-based gene vectors. All the authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.