Despite advances in systemic therapies for solid tumors, the development of brain metastases remains a significant contributor to overall cancer mortality and requires improved methods for diagnosing and treating these lesions. Similarly, the prognosis for malignant primary brain tumors remains poor with little improvement in overall survival over the last several decades. In both primary and metastatic central nervous system (CNS) tumors, the challenge from a clinical perspective centers on detecting CNS dissemination early and understanding how CNS lesions differ from the primary tumor, in order to determine potential treatment strategies. Acquiring tissue from CNS tumors has historically been accomplished through invasive neurosurgical procedures, which restricts the number of patients to those who can safely undergo a surgical procedure, and for which such interventions will add meaningful value to the care of the patient. In this review we discuss the potential of analyzing cell free DNA shed from tumor cells that is contained within the cerebrospinal fluid (CSF) as a sensitive and minimally invasive method to detect and characterize primary and metastatic tumors in the CNS. Comprehensive genomic profiling (CGP) of formalin-fixed paraffin-embedded (FFPE) tumor tissues has allowed many cancer patients to benefit from precision medicine by identifying genomic alterations for which targeted therapy or immunotherapy may be preferentially utilized. While the clinical value of tissue based CGP is well-established, this approach requires patients to undergo a tissue acquisition procedure such as a fine need aspiration, core biopsy, or surgical resection. As nearly all patients will be biopsied at presentation to establish a tissue diagnosis of malignancy, there is often tumor tissue available for CGP.Despite advancements in therapeutic strategies for solid tumors, progression frequently occurs, manifesting as metastatic disease or recurrence. At this point, the ability to acquire a second biopsy can be challenging for several reasons including, the patient's general health status, the ability and/or willingness to undergo another invasive procedure, and the patient's safety associated with the location of the lesion to be biopsied. For patients with primary brain tumors , most patients will undergo a tissue sampling procedure at the time of presentation to establish the diagnosis, while only a subset 10–20%) of patients will ever have a second procedure to acquire tissue at tumor recurrence  from the plasma of peripheral blood samples has provided a novel mechanism for interrogating the genomic landscape of tumors without invasive surgical procedures. This type of assay is frequently referred to as a “liquid biopsy” and encompasses a broad category of minimally invasive tests that can effectively isolate DNA shed from cancer cells that are circulating in the blood or other relevant fluids. In contrast to circulating tumor cells (CTCs), which are intact tumor cells in the peripheral circulation, ctDNA is a component of the total circulating “cell-free” or cfDNA that includes both ctDNA and DNA fragments in plasma contributed by white blood cells and apoptotic normal non-hematologic cells. ctDNA testing platforms range from variant-specific and “hot-spot” assays to broad next generation sequencing panels that can assess for variants in hundreds of genes simultaneously. For some solid tumors, such as breast, lung, prostate, melanoma and colorectal cancers, ctDNA assays have demonstrated strong concordance of genomic profiles compared to tissue-based assays , 4. MoreWhile advanced-stage tumors demonstrate increasing yields of ctDNA in the plasma, detecting ctDNA in the plasma of primary brain tumor patients is limited, with only a minority yielding detectable levels. Across published reports, the rate of ctDNA detection in plasma for primary brain tumors varies from 10 to 50% of samples with higher detection rates associated with glioblastoma; however, all studies consistently report that even when detected, the ctDNA concentrations are much lower compared to other advanced stage tumors , 5–7. AlSeveral studies have identified cerebrospinal fluid (CSF) as a rich and reliable source of ctDNA (ctDNA-CSF) in patients with primary and metastatic brain tumors. Secreted by the choroid plexus, CSF is a clear body fluid in the subarachnoid space of the brain and spinal cord that provides specialized immunologic protections and serves as a cushion for the brain . Given iIn this review we will explore CSF testing for primary and metastatic brain tumors and discuss the clinical utility of testing for management of patients affected by brain cancer.For tumors that arise outside of the CNS , the presence of tumor cells in the CSF correlates with poor response to therapy and overall poor prognosis , 11. To CSF is typically collected through a minimally invasive lumbar puncture but can also be accessed via existing shunts or during intracranial surgical procedures, with a typical volume of 2–10 mLs of fluid with each collection. Current methodologies for evaluating CSF focus on determining the presence of cancer cells with cytology, immunohistochemistry, or flow cytometry; however, while these techniques can be highly specific, they lack the sensitivity that is needed for early detection of malignancy. As an alternative, ctDNA from the CSF (ctDNA-CSF) can be extracted and sequenced, which increases sensitivity and provides additional information about tumor heterogeneity and progression of disease ; Figures, 2. In tHER2 amplification in the ctDNA-CSF sample but barely detectable levels in the pelleted fraction. In all, known variants were detected in 100% of ctDNA-CSF samples but only 63% of CSF cell pellets from their cohort  of ctDNA-CSF samples, however only 84.6% (22/26) and 73.1% (19/26) of pelleted CSF and plasma ctDNA samples, respectively, identified the mutation  compared to plasma ctDNA (21%). In another study of patients with leptomeningeal carcinomatosis, ctDNA-CSF analysis detected the presence of disease in 100% of cases while neuroimaging and CSF cytology identified 63 and 71% of cases, respectively. In another institution's experience, ctDNA-CSF genomic profiling from patients with malignant CNS disease detected clinically actionable mutations in 40% (11/27) of patients. After consultation with an interdisciplinary molecular tumor board, seven patients were recommended to receive a targeted therapy based on the ctDNA-CSF results, with four patients being clinically stable to proceed with the new treatment regimen. These patients included two metastatic NSCLC patients with EGFR mutations who received erlotinib or afatinib, a NSCLC patient with an EZR/ROS1 fusion who was treated with crizotinib and a HER2 positive metastatic breast cancer patient with FGFR amplification who received everolimus  only represent 1% of primary brain tumors in pediatric and young adult patients, the CNS is the second most common location for extragonadal tumors. These are a heterogeneous group of tumors that can be difficult to diagnose because of their non-specific clinical presentations and subsequently predict which tumors are more likely to metastasize to the brain. The propensity to spread throughout the CNS increases the need for highly specific and sensitive markers that can be detected in the CSF of patients. Currently monitoring certain protein biomarkers such as alpha-fetoprotein and human chorionic gonadotropin within the CSF only provides information for certain GCT subtypes and has limited sensitivity in terms of diagnosing CNS dissemination . There iPrimary CNS lymphoma (PCNSL) patients may also benefit from CSF liquid biopsies as these tumors often disseminate via CSF. Notably, surgical resection is not standard of care, and if PCNSL is diagnosed during an intraoperative frozen section, the surgical procedure is routinely stopped as this disease is best managed with chemotherapy. While this is considered best practice, it does limit the amount of tissue available for genomic testing and further reduces the likelihood of additional tissue sampling at the time of tumor recurrence. Detection of mutational profiles and immunoglobulin gene rearrangements in CSF has the potential to improve diagnostic sensitivity, especially in setting of steroid administration, which can make tissue sampling of PCNSL challenging because of tumor cell lysis. In contrast, one can speculate that the use of steroids for PCNSL prior to ctDNA-CSF liquid biopsy may enhance the amount of ctDNA available in the CSF compartment. In one study 19 patients with B-cell lymphomas were evaluated to compare the utility of ctDNA-CSF or plasma in diagnosing CNS involvement and explore whether the analysis of ctDNA could monitor treatment response in CNS lymphomas . GenomicFurther utility for CSF liquid biopsy in PCNSL includes identifying tumor recurrence, particularly in cases associated with extensive leptomeningeal involvement in which diagnosing recurrent tumor in the post-treatment setting solely by neuro-imaging studies can be very difficult, often when cytologic analysis is unrevealing. Indeed, sequential ctDNA-CSF testing in patients undergoing treatment demonstrated that despite a decrease in tumor cells by flow cytometry or in some cases no detectable disease by conventional techniques, patients still harbored CNS disease when assessed with a more sensitive method .MYD88, CARD11, TNFAIP3, and CD79B. Therefore, in the recurrent PCNSL setting, should CSF liquid biopsy identify mutations in these key genes, it would likely guide therapy away from BTKi  ibrutinib as monotherapy in salvage treatment of PCNSL, this highlights the value of mapping the genomic landscape of recurrent PCNSL. While BTKi may become an important option for salvage therapy, it is worth noting that several mechanisms of resistance have been identified including mutations in rom BTKi –33.Liquid biopsies are increasingly demonstrating clinical utility for a variety of cancers. Guidelines have evolved most rapidly in non-CNS cancers such as lung cancer where CAP guidelines suggest that liquid biopsy may be acceptable at the time of diagnosis should tissue not be available for testing.Additionally, this methodology can help clarify radiologic findings and differentiate between radiation-induced necrosis and tumor progression. Pseudoprogression, defined as post-radiotherapy changes that typically resolve spontaneously, occurs in 10–30% of GBM patients typically within the first 12 weeks of treatment . The abiH3F3A (K27M) mutation, it would suggest a molecular diagnosis of diffuse midline glioma, H3F3A K27M mutant, WHO Grade IV; alternatively, if the tumor harbored a KIAA1549-BRAF fusion, that would suggest a lower grade glioma such as pilocytic astrocytoma WHO Grade I. A retrospective study of 20 glioma patients demonstrated combined digital droplet PCR and targeted sequencing of known glioma associated mutations on ctDNA-CSF samples could correctly classify gliomas in 85% of cases; the only samples that failed were low-grade gliomas or tumors where ctDNA-CSF yields were low , given the anatomic location, it is often not possible to biopsy these patients and therefore they are routinely treated clinically based on imaging studies. If however ctDNA-CSF testing was available and showed a were low . ProspecThere is significant potential for ctDNA-CSF testing to disrupt the way neuro-oncology is currently practiced by providing neurosurgeons, oncologists and patients with pre-surgical, pre-radiation and pre-systemic therapy genomic diagnoses that can inform clinical management strategies. The value of total and near-total resection for high grade gliomas remains an important therapeutic and prognostic indicator. ctDNA-CSF molecular analysis has the potential to provide more information to neurosurgeons for planning surgical procedures based on genomic profiles. In instances where the tumor is based in an eloquent area of the brain or deep gray structures where resection is not possible and the purpose of surgical intervention is solely to sample tissue for diagnosis, CSF ctDNA testing may be an alternative to biopsy procedures.ERBB2 levels and other tumor-specific mutations during treatment with trastuzumab emtansine, CSF ctDNA revealed continued high levels of ERBB2, MYC copy numbers, PIK3CA, and TP53 mutations, which were identified prior to the start of treatment (H3F3A K27M levels in CSF ctDNA correlated with the presence of contrast-enhancing areas on MRI in pediatric DIPGs (As several studies have demonstrated, ctDNA-CSF sequencing can also provide opportunities for early detection of metastatic disease, which may have significant implications for therapeutic intervention. Retrospective studies have demonstrated that in the context of metastatic disease ctDNA-CSF mutations reflect CNS specific alterations, which are not identified in plasma ctDNA or extracranial metastases . Incorporeatment . This hiic DIPGs .Importantly, to fully understand the potential of ctDNA-CSF testing and incorporate the methodology into routine clinical care, prospective clinical trials are needed. Larger studies should be performed that evaluate the correlations between ctDNA-CSF, plasma ctDNA and tumor tissue samples. Future prospective studies that incorporate ctDNA-CSF testing in parallel with standard of care diagnostic and management procedures will help refine and implement the utility of ctDNA-CSF testing for patients with brain metastases and primary tumors.All of the authors contributed to the preparation and writing manuscript as each individual has specific expertise in different areas, including neuro-oncology, metastatic CNS disease, circulating tumor DNA, genomic profiling, neurosurgery, and integration of molecular data into direct clinical care.Some authors are employed by Foundation Medicine, Inc. 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.


Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus (the human herpesvirus 5) and an opportunistic pathogen that primarily infects HIV-positive and other immuno-compromised patients. Retrospective studies in the field of inflammatory bowel disease (IBD) have suggested a relationship between a concomitant colonic HCMV infection and poor outcomes in patients with an ulcerative colitis (UC) due to the presence of HCMV in surgical specimens of patients with a toxic megacolon or a steroid-resistant UC. Therefore, gastroenterologists have focused on the contribution of HCMV infections in the exacerbation of UC. Numerous studies have addressed the benefits of treating colonic HCMV reactivation in UC using an antiviral treatment. However, its clinical relevance remains uncertain as only a few prospective studies have assessed the direct relationship between clinical outcomes and the viral load of HCMV in colonic tissues. HCMV reactivation can be triggered by inflammation according to fundamental research studies. Thus, optimal control of intestinal inflammation is essential for preventing an HCMV reactivation in the intestinal mucosa. Indeed, several reports have indicated the effectiveness of an anti-tumor necrosis factor-alpha (TNFα) treatment in patients with an active UC and concomitant HCMV infections. In this review, we describe the mechanism of HCMV reactivation in UC cases and discuss the current issues regarding diagnosis and treatment of HCMV infections in UC patients. Herpesviridae, is generally contracted during childhood and can persist as a lifelong latent infection. Latent HCMV infection is reactivated under inflammatory conditions in an immunosuppressed host and can cause an organ damage [Human cytomegalovirus (HCMV), a double-stranded DNA virus belonging to the family n damage . Notablyn damage . HCMV inn damage .Ulcerative colitis (UC) is a non-specific chronic inflammatory bowel disease with ulcers and erosions in the colonic mucosa. It is thought to be caused by a multifactor involving genetic factors and environmental factors such as diet, but the etiology has not been clarified. UC mainly causes inflammation in the colonic mucosa, causing gastrointestinal symptoms such as diarrhea, hematochezia, and abdominal pain, often requiring immunosuppressive treatment.The HCMV reactivation in healthy people, which is regulated by both HCMV-specific antibodies and CD8-positive T cells, is disrupted under the immunocompromised condition. Therefore, patients with medically refractory UC could be prone to the HCMV infection because of the use of immunosuppressive drugs, especially corticosteroid in addition to sustained inflammation in colonic mucosa triggering HCMV reactivation. The association between cytomegalovirus infections and UC was first recognized over 50 years ago . The preHCMV exhibits an extensive tissue affinity in the human body and is known to infect many cells and tissues such as macrophages, vascular endothelium, epithelial cells, fibroblasts, and nerve cells . ConversContrarily, this defense mechanism is disrupted in an immunosuppressed host and HCMV is reactivated, causing damage to multiple organs, including the gastrointestinal tract. Additionally, for an HMCV reactivation, it is imperative for the virus to express the immediate early (IE) genes that trigger viral growth, followed by transcription of the early (E) and the late (L) genes that result in the formation of viral proteins. Expression of the IE gene is controlled by the major immediate-early promoter (MIEP) located upstream, with various transcription factor-binding sites in the vicinity . In partIt is difficult to distinguish clinically UC exacerbation from concomitant HCMV colitis because, typically, UC patients present with intestinal symptoms such as diarrhea and bloody stool. The various endoscopic findings are observed in UC complicated by HCMV infection. No definitive endoscopic findings lead to the diagnosis of concomitant HCMV infection in active UC. However, the existence of ulcerative lesions in UC patients refractory to immunosuppressive therapies may suggest the possibility of HCMV infection. Endoscopic findings such as punched-out ulcers, longitudinal ulcers, diffuse mucosal defect, and geographic ulcers  have beeHowever, it is clinically difficult to diagnose HCMV colitis only by endoscopic findings ,16 and iThe most reliable test method in clinical virology is isolating HCMV in culture as it proves the existence of live HCMV with no false positives or negatives related to the detection of antigens and nucleic acids. Virus isolation in culture is performed using samples such as blood, tissue, and urine . This teSerological tests are used for diagnosis by determining the changes in IgM and IgG antibody titers over time by comparing the acute- and convalescent- phase paired sera (measured after 2 weeks-4 weeks). HCMV-IgM antibody titers increase from 2 weeks to 6 weeks after the initial infection, and usually decline within 2 months to 3 months in healthy individuals. IgM antibody is rarely detected in HCMV reactivation . Recent This method detects HCMV antigen-positive cells present in peripheral blood using a monoclonal antibody against the primary structural antigen (pp65) of HCMV. Neutrophils responsible for the phagocytosis of pp65 are detected, rather than the infected cells. Quantification of positive leukocytes allows for the degree of viremia to be quantified, which reflects both clinical symptoms and disease state. However, it must be recognized that peripheral blood reactivation does not necessarily reflect HCMV reactivation in the gastrointestinal tract. Additionally, HCMV antigenemia assays have high specificity and low sensitivity for moderate/severe UC cases that are complicated by the histopathological diagnosis of HCMV colitis ,22. ChunThe HCMV-DNA test method is based on extracting a small amount of nucleic acid present in a specimen, followed by amplification and detection of DNA using DNA polymerase. Notably, the real-time polymerase chain reaction (PCR) method enables quantitative analysis and is used for monitoring HCMV infection after an organ or a bone marrow transplantation, and determining the therapeutic effect following treatment for HCMV infection. However, false positives due to DNA contamination and impedance of DNA amplification due to mutations in primer binding sites presents challenges. Blood HCMV-DNA testing is standard for monitoring HCMV infections in organ and bone marrow transplant patients, and its inclusion in the diagnosis algorithm in the UC-complicated HCMV infections has also been suggested . This meThe gold standard for diagnosing HCMV infections of each organ is the histological presence of an inclusion body. Typical HCMV infected cells are large and have cytomegalic inclusion bodies with a halo indicating an active HCMV proliferation. It is difficult to identify HCMV using only hematoxylin and eosin (HE) staining. Therefore, it is better to use immunohistochemistry (IHC) staining. The diagnostic method with histological examination affords high sensitivity (92% to 100%), but low and variable sensitivity (10% to 87%): a combination of HE and IHC staining increases the sensitivity to 78% to 93% . The senThe treatment of HCMV infection in patients with active UC is complicated by the difficulty experienced in exactly distinguishing an HCMV reactivation from HCMV colitis, as inflammatory conditions in the colonic mucosa of UC patients may contribute to an HCMV reactivation ,31. WhenThe HCMV antigenemia assay is widely used for decision of initiating antiviral treatment. However, the use of HCMV antigen tests as diagnostic tools for HCMV colitis remains unclear. In clinical practice, we often encounter that the HCMV antigen is not detected even if HCMV infection in patients with active UC is suspected. Matsuoka et al.  reportedAs mentioned above, the histological test confirming the presence of HCMV using HE or IHC staining is the gold standard for diagnosis. Therefore, if the histological examination shows the existence of HCMV in colonic mucosa, antiviral treatment should be considered, although it may depend on the number of HCMV-positive cells.3 copies/105 cells in diseased mucosa in comparison to non-diseased mucosa, while non-refractory patients and control displayed levels below this threshold, thus allowing HCMV colitis to be distinguished from mucosal infection. Also, Okahara et al. [In HCMV-DNA analysis, the initiation of antiviral treatment is considered for patients with a high viral load in colonic mucosa ,28. Howea et al.  suggestea et al.  reportedThe effects of antiviral treatment on UC with HCMV colitis have been observed in a few studies. Recently, in a meta-analysis of 15 studies, stratified analysis focused on cases of steroid resistance showed that the colectomy rate was significantly lower in the group treated with antiviral treatment than in the control group . Jones eIn UC cases that are complicated with HCMV infection, suppression of HCMV reactivation by anti-inflammatory treatment is also important. The risk of complications accompanied by HCMV infection is high when steroids and cyclosporine are administered due to strong suppression of the immune system of the host ,41,42. EAdditionally, we have reported on the effectiveness of granulocyte/monocyte adsorptive apheresis (GMAA) for UC-concomitant HCMV colitis. It appears that the GMAA has little effect on HCMV reactivation, because the GMAA does not directly affect the local immune system. It may be considered as an induction treatment to UC patients with a history of an HCMV infection ,46. ConsIn recent years, the evolution of biological products has been remarkable, and new treatments suppressing the HCMV reactivation will appear.It has been hypothesized that IBD cases concomitant with CMV infections have a poor prognosis. However, the exact mechanism by which HCMV exacerbates IBD remains unclear. We had previously reported an experimental IBD mouse model that was exacerbated by a CMV infection ,51. The Reactivation of HCMV causes exacerbation of disease activity of UC patients. The diagnosis continues to be based on HCMV detection in histopathology, but we believe that the quantitative PCR method is a promising alternative approach to better define the extent of an HCMV reactivation in histological negative cases. The indications for antiviral therapy in IBD remain controversial, but more recent findings suggest the benefit of antiviral treatment in patients with a histologically proven HCMV colitis or a high HCMV-DNA load in colonic tissues determined by quantitative PCR. In addition, insufficient control of inflammation can reactivate HCMV, resulting in the increasing disease activity of the UC. Therefore, optimal control of colonic inflammation must be achieved in UC patients positive for HCMV infections who are resistant to conventional immunosuppressive treatment. Further experimental data and prospective studies will be required in the future to develop appropriate therapeutic approaches for active UC patients complicated with HCMV infection.


Sickle cell disease (SCD) is an inherited blood disorder that predominantly affects individuals in sub-Saharan Africa. However, research that elucidates links between SCD pathophysiology and nutritional status in African patients is lacking. This systematic review aimed to assess the landscape of studies in sub-Saharan Africa that focused on nutritional aspects of SCD, and highlights gaps in knowledge that could inform priority-setting for future research.The study was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Inclusion criteria comprised original, peer-reviewed research published between January 1995 and November 2020 involving individuals in Africa with any phenotypic variant of SCD and at least one nutritional status outcome. Nutritional status outcomes were defined as those that assessed dietary intakes, growth/anthropometry, or nutritional biomarkers. Databases used were Ovid Embase, Medline, Biosis and Web of Science.The search returned 526 articles, of which 76 were included in the final analyses. Most investigations (67%) were conducted in Nigeria. Studies were categorized into one of three main categories: descriptive studies of anthropometric characteristics (49%), descriptive studies of macro- or micronutrient status (41%), and interventional studies (11%). Findings consistently included growth impairment, especially among children and adolescents from sub-Saharan Africa. Studies assessing macro- and micronutrients generally had small sample sizes and were exploratory in nature. Only four randomized trials were identified, which measured the impact of lime juice, long-chain fatty acids supplementation, ready-to-use supplementary food (RUSF), and oral arginine on health outcomes.The findings reveal a moderate number of descriptive studies, most with small sample sizes, that focused on various aspects of nutrition and SCD in African patients. There was a stark dearth of interventional studies that could be used to inform evidence-based changes in clinical practice. Findings from the investigations were generally consistent with data from other regional settings, describing a significant risk of growth faltering and malnutrition among individuals with SCD. There is an unmet need for clinical research to better understand the potential benefits of nutrition-related interventions for patients with SCD in sub-Saharan Africa to promote optimal growth and improve health outcomes. Sickle cell disease (SCD) is the most common inherited blood disease worldwide, with the vast majority of cases occurring in sub-Saharan Africa . The conIn high resource countries, mortality from SCD has decreased dramatically over the past five decades. The improvements in outcomes have been attributed in part to newborn screening and comprehensive care programs designed to prevent disease complications to the extent possible and to treat complications of disease when they occur . Early dThere is evidence that the pathophysiology of SCD has substantial nutritional implications including higher energy and nutrients requirements, nutrient deficiencies, and growth abnormalities –15. It iWe sought to include all studies involving original research that focused on the nutritional status of individuals with SCD in an African population. Studies involving nutritional status were defined as those that investigated topics of dietary intake, measurements of growth or anthropometry, or nutrition-related biomarkers. Studies that did not differentiate the cause of the anemia were excluded, as were studies that only included nutrition interventions as part of comprehensive care programmes . Studies involving both children and adults were included. The focus of this analysis was on studies involving individuals with various forms of SCD including HbSS, HbSC, and rarer genetic variants of disease; studies were excluded that only involved individuals with sickle cell trait. Case reports and review articles were also excluded.The databases employed for this search were Ovid Embase, Medline, Biosis, and Web of Science. The date range was January 1st, 1995, through November 30th, 2020, such that the reference list covered a period of approximately 25 years. We performed a Boolean search using specific Boolean operators and the following search terms: “Sickle cell disease” or “sickle cell anemia” or “hemoglobinopathy” AND Africa or specific African countries  AND various nutrition-related terms  along with associated terms (both indexed and non-indexed) for nutrition, diet and growth, and specific vitamins and minerals. We also allowed for inclusion of articles that were identified through review of the bibliographies of papers that underwent full-text review. Investigations written in any language were included provided sufficient translation into English could be assured.Titles and abstracts were each screened by two independent reviewers. Any title or abstract that appeared to meet inclusion criteria or for which there was uncertainty prompted a full text review. Reviewing of full text articles were assigned to individual investigators. If eligibility of a full text article was unclear, it was resolved by discussing it with at least two other reviewers on the research team who were not earlier assigned the full text article using the inclusion criteria. To maximize consistency among reviewers, each reviewer initially reviewed 10 articles and the review team together discussed the initial dataset that had been extracted to ensure accuracy and completeness. The review process then proceeded according to the process described.Study data were extracted into standardized forms using Microsoft Excel  where they were organized for analyses. Depending on the nature of the articles that met inclusion criteria, we extracted information relating to geography, subject age group, sample size, comparison groups, biomarkers, and other relevant variables. Since the main purpose of this investigation was to broadly understand the types of nutrition-related studies that have been conducted involving individuals with SCD in Africa, we chose not to systematically judge the quality of evidence or risks of bias within individual studies. Rather, we discussed specific merits and limitations of individual studies where appropriate in the context of major themes that would emerge in the analyses. We planned for a quantitative categorization of the types of articles  and a narrative synthesis of data in table and text format to summarize and assess the results.The study was conducted and reported according to PRISMA  guidelines , 21.In total, 526 unique titles and abstracts were identified through the literature search. Of those, 347 did not meet inclusion criteria and 179 full-text articles were assessed. Seventy-six studies were deemed eligible and included in the final analyses  were conducted in a single country (Nigeria). Each study was placed into one of three main categories according to the primary nature of the investigation: (a) descriptive study of anthropometric characteristics ; (b) descriptive study of macro- or micronutrient status ; and (c) interventional studies . The studies are summarized in Tables Nearly one-half of all studies identified focused on anthropometric characteristics. The studies typically assessed height, weight, and body mass index (BMI). Other measurements included head circumference, arm span, and various body composition parameters.The majority 25/37; 68%) of studies were conducted in Nigeria. Other studies involved populations in Democratic Republic of Congo (DRC) , 50, 57,; 68% of The three largest studies found significant growth defects compared to healthy controls. A multi-country study of more than 3500 SCD patients (aged 10–24 years) with nearly 1000 controls in Cameroon, Ivory Coast, Gabon, Mali, and Senegal was designed to evaluate determinants of vascular complications . AnthropThe studies involving smaller sample sizes showed greater variation in the results, but some trends emerged. Several reports confirmed the finding that males were more likely to show growth defects than females , 29, 45.The second most common group of studies identified involved assessment of biomarkers from serum samples for macronutrients or micronutrients. Most studies were conducted in Nigeria ; other studies took place in DRC , Tanzanin = 11) compared with those who were alive at the end of the study period. Those who suffered mortality had a significantly lower BMI, plasma taurine levels and arginine bioavailability before succumbing [Serum protein levels were investigated in one small study  in Nigeria in which no significant differences were reported in the concentrations of total protein or albumin between SCD patients and controls . Howeverccumbing .Proportions of fatty acid and the state of metabolism were evaluated in four related studies of young SCD patients in Nigeria , 60, 61.Acknowledging that interpretation of plasma concentrations of vitamins and minerals can be problematic in patients with ongoing inflammation, analysis of serum vitamin levels in SCD patients generally indicated lower concentrations of vitamin A , 65 vitaSelected minerals were evaluated in eight small studies of SCD patients and compared to healthy controls. Serum iron concentration was reduced in patients compared to controls in all studies that evaluated it –76, 82. n = 65) as well as controls (n = 60) were given folic acid, vitamin B complex, and proguanil, with the treatment group also given twice-daily oral lime juice with weight-based dosing ranging from 5 to 15 mL. Each child was assessed monthly for 6 months. The group receiving lime juice was reported to have significantly fewer pain episodes, febrile illnesses, and hospital admission rates. There was no change in transfusion rate, organomegaly, or jaundice. The positive effect was postulated to result from vitamin C, amino acids , and flavonoids contained in the juice, but no direct evidence for this was provided [A very small number of clinical studies involving nutritional interventions in SCD patients in African countries were identified. There were four randomized trials –91. The provided .. The hypothesis was that omega-3 fatty acids could reduce red blood cell aggregation, adherence, and inflammation that occur during sickle cell disease-mediated vaso-occlusive crises. The treatment group had significantly fewer clinical vaso-occlusive events, as well as reduced rates of severe anemia and need for blood transfusions. The study was not powered to detect changes in other outcomes such as stroke, sequestration crisis, or vascular necrosis [A second study of 128 SCD patients aged 2–24 years in Sudan investigated the impact of 1 year of treatment with oral omega-3 capsules containing EPA and DHA fatty acids (using age- and weight-dependent dosing) compared to placebonecrosis .A third trial utilized a double-blind, random order design. Ready-to-use-supplementary food was studied in 119 children with SCD in Tanzania . Two difThe final randomized study identified involved the regular administration of oral arginine therapy to 35 hospitalized patients with SCD in Nigeria and compared the effects with 33 control subjects. Plasma arginine levels increased by 125% in the arginine arm compared with 29% in the control arm . ArgininOne of the non-randomized interventional studies identified investigated the use of vitamin D supplementation. A small treatment arm was nested in a Nigerian study comparing blood levels of vitamin D and pro-inflammatory cytokines . The hypTo our knowledge this is the first review of nutrition-related studies involving individuals living with SCD in sub-Saharan Africa. While a moderate number of studies were identified, most were descriptive in nature and small in terms of numbers of subjects. Approximately two-thirds of studies took place in a single country (Nigeria). In addition, there were very few interventional trials designed to measure the impact of an isolated nutritional intervention and only four randomized studies. The findings of this review suggest an outstanding need for nutrition-focused research relating to the care of individuals with SCD in Africa, with a particular emphasis on research with practical implications for clinical management in order to improve patient outcomes.The findings of studies identified through this review are generally consistent with nutrition-related investigations involving SCD patients in other parts of the world. More than 50 years ago, poor growth was first reported in patients with SCD, and that observation has since been repeated in multiple studies involving SCD populations in Jamaica, Brazil, India, and North America , 93–99. Evidence derived from robust interventional studies is important to support recommendations for specific nutrition-related practices for patients with sickle cell disease. Only four randomized trials were identified. The studies were small, each involving less than 150 individuals with SCD. Positive clinical benefits were found with the use of lime juice, long-chained fatty acid supplementation, RUSF, and oral arginine; ideally these findings would be confirmed in larger follow-up investigations. It is worth noting the paucity of robust interventional trials designed to test the effect of macronutrient supplementation in individuals with SCD despite the evidence, as described above, that nutrition deficits in this population are likely to be caused at least in part from the increased energy demands that result from altered metabolism.Guidelines for clinical management of patients with SCD published by internationally recognized organizations do not provide special guidance for nutritional care , 106. GiLimitations of this systematic review include the fact that nearly half of studies identified were largely anthropometry-based descriptive studies. Few studies involving nutrition interventions in sub-Saharan Africa were identified, only several had robust methodologies, and none have been validated in repeated studies. In addition, the studies involving analyses of vitamin and mineral levels in SCD patients in sub-Saharan Africa overall involved small numbers of patients and generally were unable to link findings with meaningful clinical correlations in ways that might influence nutritional care practices. Another limitation is that most investigations identified took place in the single country of Nigeria .Despite the reality that most SCD patients globally live in sub-Saharan Africa, and the fact that nutritional disturbances in SCD patients are increasingly well described, there has been limited research focused on ways that nutritional care might help to improve clinical outcomes in this patient population. A systematic review of the literature revealed studies that consistently reported stunted growth and malnutrition in African SCD patients during childhood and adolescence, but failed to identify robust, validated studies that could be used to inform clinical management. Our study suggests an outstanding need to determine if and how supportive nutritional care can reduce disease severity and improve health outcomes for individuals with SCD in sub-Saharan Africa. As such, priority research in this area in the future may include systematic assessment of the drivers of nutritional status in SCD patients, studies that directly advance the understanding of macro- and micronutrient deficiencies associated with clinically significant physiologic effects, and investigations that evaluate the impact of nutritional interventions to inform evidence-based nutrition guidance.


Nonalcoholic steatohepatitis (NASH) progresses from nonalcoholic fatty liver disease (NAFLD), which is common in the elderly and is an important predictor of liver failure and hepatocellular carcinoma (HCC). NASH also has a robust and multifaceted relationship with aging-related diseases such as metabolic syndrome, cardiovascular diseases, and diabetes. Due to older people’s activity limitations, pharmacological treatment is essential, but an incomplete understanding of NASH pathophysiology limits drug development. Cholesterol (low-density lipoprotein) has been identified as a cardiovascular disease risk factor for a few decades. Recently, liver free cholesterol was proven as a NASH inducer .in vivo and in vitro due to inhibited degradation through cholesterol-blocked Hippo activity. They also described a signaling pathway that includes cholesterol transporter AsterB/C, soluble adenylyl cyclase (sAC), PKA, and IP3R. Via this pathway cholesterol is transported into cytoplasm and mediates downstream calcium signaling to induce RhoA activity and suppresses Hippo kinase-Lats1/2, leading to increased TAZ and YAP (another TAZ homologue in the Hippo pathway) protein at the post-transcriptional level. The elevated TAZ triggers the fibrosis and induces NASH [In human NASH liver, there are increases in both cholesterol and TAZ  protein, yet the molecular mechanism linking cholesterol to TAZ upregulation in NASH remains undetermined. TAZ is one of the transcriptional regulators in the Hippo pathway that play roles in development, organ size and homeostasis, and carcinogenesis. TAZ is negatively regulated by upstream signaling: MST1/2 kinases activate LATS1/2 kinases to phosphorylate TAZ, suppressing its activity by proteasomal degradation. Dephosphorylated TAZ translocates into the nucleus, where it interacts with TEAD to activate specific target gene expression. A dedicated study by Wang et al. [in vivo and in vitro: TAZ in hepatocytes induces the synthesis and secretion of Indian hedgehog (Ihh), and Ihh activates hepatic stellate cells to promote fibrosis. The study further introduced an important new mechanism to understand the role of cholesterol-induced TAZ in hepatocytes in animal models with corroborating evidence in human NASH, and suggested TAZ as a plausible therapeutic target in NASH. Although there are safety concerns about targeting TAZ specifically in hepatocytes, lowering TAZ properly in chronic disease should be feasible since TAZ expression in the healthy liver is very low or undetectable compared with NASH liver.In another issue of Cell Metabolism, Wang et al.  investigAfter the first hepatocyte-targeted siRNA was approved for clinical use, hepatocyte-specific protein, TAZ, was targeted by the stabilized GalNAc-siRNA in NASH . The resIn summary, the findings of Wang et al.  have ide


Gene therapy is a therapeutic strategy of delivering foreign genetic material (encoding for an important protein) into a patient’s target cell to replace a defective gene. Nucleic acids are embedded within the adeno-associated virus (AAVs) vectors; however, preexisting immunity to AAVs remains a significant concern that impairs their clinical application. Extracellular vesicles (EVs) hold great potential for therapeutic applications as vectors of nucleic acids due to their endogenous intercellular communication functions through their cargo delivery, including lipids and proteins. So far, small RNAs (siRNA and micro (mi)RNA) have been mainly loaded into EVs to treat several diseases, but the potential use of EVs to load and deliver exogenous plasmid DNA has not been thoroughly described. This review provides a comprehensive overview of the principal methodologies currently employed to load foreign genetic material into EVs, highlighting the need to find the most effective strategies for their successful clinical translation. Nucleic acid-based therapies are rapidly evolving in preclinical and clinical trials for several genetic diseases ,2,3. VirThe limitations of AAV vectors have a significant impact on their complete clinical translation into human diseases. First, the time-consuming conversion of single-stranded to double-stranded AAV genomes can delay the onset of transgene expressions . The virThe other main hindrance associated with AAV vectors is their limited genome packaging capacity (~4.5 kb). For the packaging of foreign DNA molecules into AAV capsids (WT-AAV genome or any transgene cassette), the DNA sequence must be flanked by the cis-acting ITRs. These represent the packaging signals and allow the amplification of the DNA genome by a self-primed replication mechanism. The replication products are DNA genomes of positive and negative polarities packaged into the AAV virions with equal frequencies . EffortsThe study on exosomes is continually growing, to yield valuable information regarding their intrinsic properties in regulating complex intracellular pathways, including those underlying neurological disorders . In contHowever, the most important questions still need to have answers: How and where are misfolded proteins packaged into exosomes, and how can exosomes be recruited by recipient cells? Continued research of the complex architecture of exosomes will provide insights into how these vesicles may be therapeutically targeted in the future.Heterogeneity is a critical aspect in the exosomes field, given that it represents an underlying factor that has an impact on current and novel exosomes isolation techniques. Exosomes are membrane-bound EVs released from cells into the extracellular space . ExosomeDue to these functions, exosomes have brought to challenge the traditional classification of enveloped and nonenveloped viruses , providiIn comparison to virus-derived vectors, exosomes have several advantages: mainly their cost-effectiveness, availability, and reduced risk of immune responses but, also, almost unlimited transgene sizes and the possibility of repeated administrations. However, due to the small sizes of exosomes, the capability to encapsulate plasmids with varied sizes is still a matter of debate of the ongoing research; thus, several strategies have been reframed to address this issue, as listed in Transfection is the procedure of a non-viral-mediated delivery of foreign genetic material into host cells . DependiThe electroporation approach seems to guarantee a more reliable encapsulation of foreign genetic material, maintaining EVs integrity and functionality, and seems to be a viable alternative for cell types that are not responsive to the transfection method. An electrical pulse at an optimized voltage (1° C/pulse) has been applied to not damage the EVs membrane and overcome the cell membrane capacitance, disturbing the phospholipid bilayer of the membrane and creating transient membrane pores through which charged molecules like DNA cross . LamichhThere is another method for DNA loading in EVs, such as sonication ,108. SonThe discrepancies emerged concerning the content of different part-genomic DNA (gDNA)  and carrAn enigma links plasmid DNA and AAV vectors, respectively: DNA-associated persistence and the persistence of the AAV genome. Plasmids are ubiquitous in nature; whereas plasmid DNA can be transmitted and replicated in the bacterial cell , chromosomal DNA can only vertically be transmitted between daughter and mother nuclei . HoweverAn additional important point regarding exogenous DNA loading has not been thoroughly addressed in this review: the different cellular responses induced by the transfected DNA. As mentioned above, the introduction of plasmid DNA into mammalian cells is not fallowed by its complete integration inside the genome, but it persists as extrachromosomal DNA for a period. Despite that introduced plasmid DNA is subjected to degradation, some of it can acquire a nucleosome structure; a study conducted by Igoucheva et al.  reportedConsiderable interest is addressed in developing nonviral lipid-based vectors due to their potential to load and transfer a large amount of DNA into cells. Cationic-lipid (CL)-based vectors are synthetic carries of nucleic acids currently employed in gene delivery, like CL-DNA , and genExosomes have many similarities with liposomes ; this haInspired by biological cells’ architecture, considerable attention has been paid to assembling multicompartment systems using a biomimetic approach in the last decade. The field of biomimetics has opened a new frontier of a wide range of biomedical applications aimed at improving patient compliance with low system side effects. Considerable progress has been achieved in reconstructing and assembling multicompartment systems based on liposomes composed of self-assembled bilayers of phospholipids, the active compound of the architecture of cell membranes that makes them advantageous in terms of a lack of toxicity, biodegradation, and biocompatibility ,139.Due to their internal architecture of phospholipid bilayers, two different types of liposomes can be distinguished: unilamellar and multilamellar. Unilamellar liposomes consist of a single phospholipid bilayer that forms a physical barrier between the internal compartment (aqueous core) and the external environment, with particle sizes ranging from 0.1 to one micrometer. Instead, multilamellar liposomes are formed by several concentric phospholipid bilayers, with particle sizes until 100 micrometers . SeveralEven though ongoing research is focused on optimizing the purification and analytical procedures for the study of EVs, the loading of plasmid DNA keeps being limited to a particular size of nucleic acids. Likely, a deeper understanding of the biological origin of DNA inside EVs might be relevant in enhancing further methodologies of loading, making EVs-efficient foreign DNA delivery systems.


Mitochondrial DNA (mtDNA) is a circular genome of 16 kb that is present in multiple copies in mitochondria. mtDNA codes for genes that contribute to mitochondrial structure and function. A long-standing question has asked whether mtDNA is epigenetically regulated similarly to the nuclear genome. Recently published data suggest that unlike the nuclear genome where CpG methylation is the norm, mtDNA is methylated predominantly at non-CpG cytosines. This raises important methodological considerations for future investigations. In particular, existing bisulphite PCR techniques may be unsuitable due to primers being biased towards amplification from unmethylated mtDNA. Here, we describe how this may have led to previous studies underestimating the level of mtDNA methylation and reiterate methodological strategies for its accurate assessment. The existence of mitochondrial DNA (mtDNA) methylation has been controversial for decades (reviewed in ). Howeveet al. [The most widely used technique for the study of mtDNA methylation is targeted/locus-specific/amplicon bisulphite sequencing , 16–19. et al.  suggesteet al. , 13.et al., the forward and reverse primers used to assay the MT-COX1 gene region in mtDNA contain four and three non-CpG cytosines, respectively (Another reason that may make targeted bisulphite sequencing a less accurate method for measuring methylation in mtDNA is the inherent bias of these assays when a DNA template has high levels of non-CpG methylation . As DNA ectively , which wUnlike most mammalian nuclear genomes, non-CpG methylation is abundant in plant genomes. Accordingly, plant researchers have proposed methods  to allowA further risk of degenerate primers is that the introduced variation in primer sequence increases the possibility of non-specific amplification. If the mtDNA purification procedures do not adequately exclude the nuclear genome, degenerate primers increase the likelihood of amplifying mtDNA-like sequences from nuclear DNA . One solThe unique challenges for analysing the epigenetic state of mtDNA have, throughout the development of the field, inspired innovative work to surmount those challenges. In In summary, given the rapidly increasing interest in the field of mtDNA methylation, more research is urgently required to investigate the underlying mechanisms and the functional role of methylation in development, environmental responses and disease. Targeted bisulphite sequencing has proven to be a quick and reliable method for assaying methylation levels in the nuclear genome. However, the discovery of widespread non-CpG methylation in mtDNA, means that adaptations to currently used methodologies are required to make targeted bisulphite sequencing a viable tool for researching this unique genome. Finally, as non-CpG methylation is present at significant levels in the nuclear genome in the brain and pluripotent cells , the appConflict of interest statement. None declared.


Excitingly, Acomys species demonstrate scar-free healing in a wide-range of tissues beyond the skin. In this perspective article, we discuss published findings from a variety of tissues to highlight how this emerging research organism could shed light on numerous clinically relevant human diseases. We also discuss the challenges of working with this emerging research organism and suggest strategies for future Acomys-inspired research.The spiny mouse  To address this, the field of regenerative medicine has emerged with a hope to replace and restore the structural and functional integrity of the damaged tissue for individuals suffering from debilitating conditions.Damaged tissues or organs, especially in adult mammals, do not regenerate and are instead replaced by a dense scar through processes collectively known as fibrosis. Chronic fibrosis can affect tissues throughout the body, ultimately leading to organ failure and death. Annually, millions of people worldwide lose their lives to fibrosis and, in the United States, fibrosis-related deaths account for approximately 45% of all deathsAcomys2) have emerged as an exciting new organism for research in regenerative medicine. Several species of Acomys are typically found in dry-arid conditions of the Middle East, South Asia, and parts of Africa, and local populations traded stories of Acomys species’ autotomic tail “degloving” for generations. The legend of Acomys was perpetuated globally as certain species became attractive exotic pets, and the regenerative capacity of Acomys was first experimentally documented by Seifert et al.  compared to other common rodents12. Given these unusual features of Acomys biophysiology that often parallel humans, the remarkable ability of Acomys to undergo scarless regeneration makes them all the more attractive as a research organism for regenerative medicine. In this perspective review article, we discuss recent findings of Acomys regeneration and describe opportunities and challenges of future research in the hopes of expanding related research and, ultimately, helping patients recover from a wide range of acute and chronic conditions.Observational reports of field-caught Acomys regeneration have come from dermal wound healing studies that include full-thickness biopsy punches of the skin and ear and thermal burns of the skin16. Dermal fibrosis in humans can occur as a result of an accidental injury, life-saving surgery, or systemic fibrotic disease like scleroderma, severely affecting the functionality of the damaged area and the overall well-being of the patient. While progress has been made in mitigating scar tissue, complete restoration  of skin with intact hair and glands remains a clinical challenge. After injuries mimicking human trauma, Acomys skin undergoes rapid re-epithelization and regrows with hair follicles, dermis, glands, and muscle through the continued proliferation of cells17. In addition to full-thickness skin regeneration, ear holes in Acomys made via full-thickness biopsy punch fully close with newly formed blood vessels, cartilage, muscle, and nerve fibers occupying the regenerated region4. Though closure of ear hole punches has been well documented in rabbits and a select strains of immunodeficient mice20, most mouse species, including one sympatric with Acomys, do not regenerate after ear punches14. 2 mm ear hole punches has been reported to close in Murphy Roths Large (MRL/MpJ) mice, a strain known as “super healers”, but they do not fully close large (>4 mm) ear holes14 nor do they heal full-thickness excisional wounds22. Similarly, FOXN1-deficient (nude) mice heal incisions without scarring18, but regenerative properties, e.g., after large wounds, have not been characterized to date. Regeneration of large dermal wounds via extensive proliferation of multipotent cells remains unique to Acomys among adult mammals, underscoring the utility of Acomys as a research organism to investigate mechanisms underlying dermal wound healing.Early insights into Acomys skin regeneration remain elusive, initial comparative studies of Acomys and Mus, a standard laboratory mouse, suggest that immune cells play a central role in the orchestration of scar-less regeneration of Acomys skin and ear wounds. Following an injury, pro-inflammatory factors are downregulated and pro-reparative factors are upregulated in the Acomys wound bed compared to Mus23. Though inflammatory macrophages (M1-type) are minimal or absent in healing Acomys wounds, experimental depletion of all macrophages delays ear hole closure in Acomys15, reminiscent of failed regeneration in axolotl24 and mouse digit-tips25. Such experiments suggest that impaired regeneration may be related to the depletion of pro-reparative macrophages (M2-type) that are otherwise abundant in Acomys wounds15. Acomys also have lower neutrophils in comparison to Mus26, suggesting differences in other immune cell populations and function. Future Acomys studies including baseline characterization of tissue-specific immune cells and their relative contributions to various injury models could provide clinically-relevant insights that support intervention strategies to treat and manage fibrotic diseases.While the mechanisms behind Acomys skin and characterization of immune cell responses have inspired researchers to explore a broad range of questions in regenerative medicine, including musculoskeletal, nervous system, cardiovascular, and renal regeneration.Demonstration of scar-free regeneration of 27. Musculoskeletal injuries often happen in conjunction with dermal wounds after trauma and can significantly impair a patient’s mobility, leading to an increased risk of chronic health conditions. While preclinical studies from small laboratory animals, such as rodents, and large farm animals, such as goats, pigs, sheep, and cows, have improved our fundamental understanding of disease pathophysiology, challenges in functional restoration of skeletal muscle have limited progress towards improving patient health and well-being.Musculoskeletal conditions such as volumetric muscle loss injury, sarcopenia, and osteoarthritis are the single highest contributor to global disability worldwide, with nearly a quarter of the world living with a painful musculoskeletal conditionAcomys. For example, the tibialis anterior regenerates in Acomys at a faster rate than in Mus after a single injection of myotoxin, a peptide found in snake venoms leading to muscle necrosis17. Further, after repeated injections of myotoxin, Mus failed to regenerate and replaced myofibers with adipose cells instead, whereas Acomys repeatedly regenerated in a consistent fashion, demonstrating its superior regenerative properties  can be fully repaired with functional reconnection of the rostral and caudal parts of the injured spinal cord29, possibly because glial cells support a regenerative state30. The adult zebrafish (Danio rerio) also shows the ability to regrow the brain, spinal cord, and retinal tissue from resident glial cells following lesions32. However, to date, there has been extremely limited success with preclinical testing of mammalian regeneration strategies gleaned from these non-mammalian models.Functional recovery after damage to the mammalian central nervous system (CNS) notoriously presents a profound challenge to clinicians due to the remarkably poor capacity of neurons to regenerate, and an estimated 80 million individuals globally are living with disability from traumatic brain and spinal cord injuriesAcomys has emerged as an up-and-coming experimental mammalian model for CNS regeneration. Following a spinal cord injury, Acomys showed a very unique molecular and immunohistochemical response in the injured spinal cord and more rapid recovery of bladder function as compared to the commonly studied laboratory mouse . The unique responses included significantly increased expression of neurogenesis-related genes, as well as reduced histological evidence of fibrosis , and more actively proliferating cardiomyocytes were present in Acomys than in Mus40.Cardiovascular disease remains the leading cause of death in the United States and is increasing globallyAcomys may provide clues to avoiding hypoxic damage altogether. Non-traditional research organisms such as the naked mole-rat have previously proven useful to study mechanisms of low oxygen (hypoxia) in mammals41. In addition, injecting stem cells can improve cardiac function in Mus post-injury by activating an acute immune response as characterized by the induction of CCR2+ and CX3CR1+ macrophages42. If the innate immune system of Acomys can effectively regulate inflammation post-injury, understanding these mechanisms of Acomys regeneration may offer insight into the prevention and reversal of heart disease.In addition to understanding pathways that control cardiomyocyte proliferation, Acomys are intriguing, additional work is necessary to unravel the mechanisms underlying adult mammalian regeneration for eventual translation into clinics. We foresee limited availability of Acomys (species and numbers) for the broader research community as a major hurdle for widespread research applications. To date, Acomys are not maintained by vendors such as Charles River or the Jackson Laboratory; rather, small colonies are maintained by a handful of researchers for their individual projects. Procurement of Acomys is currently done by agreements between individual researchers and their institutions, limiting the widespread use of Acomys among researchers with diverse research interests. With growing enthusiasm for studying Acomys, a strategic plan to establish and disseminate various Acomys species 2 in large numbers, including the three species  that have been proven to regenerate, would significantly benefit the biomedical research community.While recent findings in Acomys colonies require adjustments from traditional processes for laboratory rodents. Compared to regular laboratory mice, Acomys take longer to reach sexual maturity (2–3 months of age), have a more extended gestational period (approximately 40 days), and produce small litters of 1 to 3 pups, all of which make it difficult to maintain and build a colony with a large number of animals. Housing conditions that increase breeding also differ from standard mouse protocols, including extra space, a protein-rich diet, additional environmental enrichment, and a higher temperature (~26 °C). In addition to environmental considerations, Acomys have weak skin and tail autotomy, so researchers must adopt special handling and restraining techniques for routine husbandry practices, injections, and behavioral assessments43. Example modifications include (a) the use of hand towels to restrain for identification of sex, subcutaneous injections, and ear tattooing, (b) the use of plastic restraint bags while collecting bodily fluids such as urine and measuring vitals such as body temperature, and (c) the use of plastic cups for transferring. Additional considerations for Acomys care and husbandry are nicely discussed in recent reviews45.Maintaining Acomys species for observational studies dates back to the early 20th century, modern demonstrations of its unique ability to regenerate a range of adult tissues have reinvigorated enthusiasm for Acomys as a research organism for a wide range of clinically relevant diseases. One of the features potentially underpinning Acomys regeneration is an altered immune response. Previous studies across a variety of injury models suggest that a blunted inflammatory response in the Acomys wound bed facilitates regeneration46; however, the exact role, timing, and interplay of immune cells, including various macrophage phenotypes, are not well understood. Cultures of primary cells in combination with transgenic approaches to knock down or silence genes of interest are likely to yield insights more quickly than whole-organism modification, given the complexities of Acomys reproductive biology. In vitro experiments also afford scalability for higher throughput assays to investigate mechanistic and functional implications of a particular signaling pathway. Since stromal cells from Acomys also exhibit unique anti-fibrotic behavior47, testbeds to isolate independent functions of specific cell types could be advantageous for translational applications. Interactions among multiple cell types, such as the interplay between parenchymal, immune, and stromal cells, could be parsed out using innovative co-culture testbeds. Taking a step further, in vivo systems such as engineered chimeras and transgenic mouse lines could also be useful to unravel underlying cellular and molecular mechanisms of mammalian regeneration.Although the use of Acomys studies. As its common name suggests, spiny mice were initially grouped under the Murinae subfamily with regular mice and rats; however, it is becoming increasingly apparent that Acomys “mice” are not the same as traditional laboratory mice and rats. In practice, this distinction is impactful because molecular reagents and resources developed for common rodent research organisms often do not work well on Acomys species. Empirical evidence that surface markers used for immunophenotyping mice and/or human cells fail to react with Acomys cells49 is supported by molecular and evolutionary findings from the 1990s, providing evidence of Acomys’ proximity to the Gerbillinae subfamily that comprises Mongolian gerbils51 and other specialized East African rodents52. Therefore, to draw helpful conclusions from Acomys-related studies, additional control experiments, along with careful validation of existing molecular tools, are warranted. Furthermore, efforts should be made towards developing Acomys-specific molecular reagents.In addition to the challenges associated with breeding and colony maintenance discussed in section 3, there are methodological challenges to take into consideration before commencing Acomys genome information. Several recent studies have resulted in RNA-Seq transcript sequence collections from Acomys to explore gene content33 and differential gene expression within Acomys and in comparison to non-regenerative species53. While providing a rapid and inexpensive source of expressed sequence data, short-read transcriptome assemblies are often fragmented, and transcript representation is biased by expression level and by the tissue type sampled. Using de novo assembled transcriptomes as a reference for RNA-Seq expression studies is often complicated by miss-assemblies that can underrepresent the true extent of expression for a given locus. In addition, Acomys-Mus orthologue assignments can be confounded by miss-assemblies and the inability to consult conserved synteny. To address these inadequacies and provide a foundation for future genomics analyses by the Acomys regeneration community, a whole-genome reference sequence is desirable. Toward this end, researchers at the University of Florida have recently sequenced and assembled an Acomys reference genome using PacBio long-read sequences with support from the National Institutes of Health. Approximately 60X redundant coverage of the Acomys genome (expected size ~2.3 Gb54) in PacBio reads were obtained from high molecular weight DNA extracted from the liver dissected from a single male. This reference genome is currently being annotated prior to public release in Winter 2020. This will enable the development of Acomys-specific molecular reagents and tools, thereby accelerating future Acomys studies.Furthermore, many molecular assays  are predicated on the availability of Acomys species across a wide range of tissues is intriguing, and they undoubtedly hold considerable promise from a translational standpoint. However, additional work is necessary to identify mechanisms underlying scar-less regeneration. Recent technological advancements in molecular and cell biology techniques, including CRISPR and single-cell RNA-seq, promise to advance our understanding of the regenerative power of Acomys. Supported by a growing community of researchers and innovative methods, Acomys is an increasingly powerful tool for the regenerative medicine community to leverage in its goal of restoring structure and function to damaged tissues and improving quality of life for people with devastating medical conditions.In conclusion, the regenerative capability of


Weekly counts of deaths are a key indicator of overall epidemic impact and trajectory.–Rapid mortality surveillance comprises both facility- and community-based components and depends on both data availability and transmissibility. Networks of community and health facility sites reporting deaths by age, sex and location on a daily or weekly basis provide an essential input to the outbreak response, including tracking the epidemic trajectory after adjusting public health and social measures.Facility-based surveillance may be implemented by medical records staff, observing proper infection control measures to protect themselves, including avoiding patient-care areas; observing workplace physical distancing measures; rigorously practicing hand hygiene; and use of personal protective equipment. A simple list of deaths by age, sex and usual residence transmitted weekly from a selected set of sentinel facilities is the starting point. One approach to rapid mortality surveillance is to leverage existing routine health information systems, COVID-19-specific rapid data collection platforms or existing surveillance platforms, such as integrated disease surveillance and response.Community-based surveillance may be critical where high numbers of deaths occur outside of health facilities – a situation that may worsen should health systems become overwhelmed. Community surveillance can be done by community-based health-care providers or other frontline workers whose existing remit includes the notification of vital events, provided that infection prevention measures can be observed to protect those collecting data.Ideally, with little lag-time, the system could generate weekly counts of deaths by demographic group and location. Real-time data can be compared to predicted deaths from historical data. For populations without historical data, the initial period of complete reporting can provide a baseline against which to compare emerging trends.Rapid mortality surveillance may provide policy-makers with up-to-date status reports, including spread into new areas or previously unaffected populations. This surveillance can also help target, prioritize and monitor the effectiveness of prevention and response strategies. Countries need real-time awareness of the distribution and magnitude of all direct and indirect health impacts of the COVID-19 pandemic. Establishing, scaling and improving upon rapid mortality surveillance would contribute to fulfilling this need, as well as preparing for future outbreaks.


As pluripotent stem cell-derived structures capable of self-organization and recapitulation of physiological cell types and architecture, brain organoids bridge the gap between relatively simple two-dimensional human cell cultures and non-human animal models. This allows for high complexity and physiological relevance in a controlled in vitro setting, opening the door for a variety of applications including development and disease modeling and high-throughput screening. While technologies such as single cell sequencing have led to significant advances in brain organoid characterization and understanding, improved functional analysis  is needed to realize the full potential of brain organoids. In this review, we highlight key technologies for brain organoid development and characterization, then discuss current electrophysiological methods for brain organoid analysis. While electrophysiological approaches have improved rapidly for two-dimensional cultures, only in the past several years have advances been made to overcome limitations posed by the three-dimensionality of brain organoids. Here, we review major advances in electrophysiological technologies and analytical methods with a focus on advances with applicability for brain organoid analysis.Brain organoids, or cerebral organoids, have become widely used to study the human brain  This wiAlong with genetic tools allowing researchers to characterize brain organoids and explore various mechanisms, -omics approaches have provided a much greater understanding of brain organoid development, both in healthy and disease states. Early organoids were characterized using common immunohistological markers, but in-depth characterization was limited . RecentlWhile promising, early organoid characterization left much to be desired in terms of depth—without understanding the extent of neuronal maturity and subtypes, cellular and regional interactions, and functional maturation, it is difficult to determine the usefulness of brain organoids as truly physiological models of disease . For exain vivo, resulting in host vascularization of the engrafted organoids, organoid maturation, and prolonged survival  have been increasingly adopted for screening applications and other studies due to the ability to combine the temporal resolution of patch clamping with the network resolution of calcium imaging . By analFinally, optogenetics have been employed in conjunction with the above techniques to allow for precise stimulation and mechanistic studies . OptogenWhile the electrophysiological techniques discussed above have been effective in providing functional data on brain organoids, the drawbacks of each method are notable, ultimately hindering the extent of functional analysis that can be performed. There have been many recent advances in electrophysiology that may provide improvements over these traditional methods, though these technologies are in their infancy or have yet to be applied to brain organoids. Some key advances with clear applicability for brain organoids are highlighted below and in A recently developed technique, dubbed PatchSeq, combines patch clamp electrophysiological recordings with sc-RNA-seq, allowing functional correlation to gene expression . While sOptogenetics has proven to be a useful tool for manipulation of neural activity, both in monolayer cultures and organoids. Traditionally, optogenetics has been utilized to stimulate and/or inhibit neurons of interest. Recently, the development of all-optical electrophysiology has provided a method to both manipulate and record neural activity at high spatiotemporal resolution . This mein vitro (DIV), observing similar activity as other recording methods, suggesting viability and long-term biocompatibility without sacrificing recording ability or resolution. The culture analyzed was a mixed neuron-astrocyte co-culture suspended in hydrogel, which—while less complex than organoids—demonstrates the ability to record from similar 3D cultures and neural networks, as well as to spatially map neural activity in three dimensions. A high-density 3DMEA platform may allow for more precise spatial mapping of active neurons  are currently being developed . By inco neurons . Indeed, neurons . This sa neurons , further neurons . This deAn alternative approach to obtain three-dimensional recordings of brain organoids is the use of electrodes embedded into a stretchable mesh. These “mesh nanoelectronics” can be integrated with cell monolayers in the early stages of organoid development, after which they have the ability to stretch as the organoids develop into three-dimensional structures, essentially taking the shape of the entire organoid . The uniWhile the above approaches address challenges associated with recording 3D brain organoids, considerable improvements are also being made regarding materials and electrode designs to enhance both recording capabilities and biocompatibility . Recent Bioinspired electrodes and scaffolds also have significant potential to improve recording capabilities in brain organoids . By inteFinally, in addition to the physical limitations of recording brain organoids in three dimensions, there are also challenges associated with data processing and analysis. Traditional electrophysiological data processing is typically carried out in a similar fashion, regardless of recording method. For action potential (spike) and burst analysis, recorded signals are high pass filtered, followed by spike detection and sorting, and finally parameter calculation from the analyzed spike patterns . Many laBrain organoids present enormous potential for disease modeling and understanding the human brain and represent a culmination of many advancements in stem cell biology. To improve the applicability of brain organoids, knowledge can also be taken from advances realized in other organoid models. For example, improved characterization approaches have been applied to other organoid models, such as single molecule fluorescent in situ hybridization (smFISH). This technique provides single-cell analysis while preserving spatial information, as it is performed in situ without disrupting the three-dimensional architecture of the organoids . While fin vivo models typically require overt behavioral phenotypes relevant to the diseases they are modeling, brain organoids cannot truly be considered representative of the human brain without physiologically relevant function—in this case, neural activity.Despite the significant advances and rapid evolution in brain organoid generation and engineering in recent years, reliable functional output and analysis is vital for brain organoid applications, especially disease modeling, and has lagged behind other organoid characterization, such as immunohistochemistry and transcriptomics . Just asin vivo recording, such as Neuropixels  are enabling the realization of many of these systems. Improvements in brain organoid analysis and control, such as the electrophysiological advances described in this review, could help design M-CELS with the ability to sense and process their surroundings, leading to autonomous function via neural logic and computation.Advances over the past several decades in stem cell biology, synthetic biology, and bioengineering have coalesced into a large umbrella of multi-cellular engineered living systems (M-CELS) . M-CELS,Improved recording also introduces analytical considerations. While each of these methods presents promise for brain organoid analysis, each method also presents new analytical challenges. All-optical electrophysiology requires complex image analysis, electrophysiological parameter extraction, and statistical analysis, hindering early adoption . 3DMEAs Ultimately, the approaches highlighted here represent a new wave of brain organoid functional analysis. By incorporating these with existing organoid technologies, such as optogenetics and sc-RNA-seq, researchers can extract more valuable information from brain organoids . The rapAP and SS designed, wrote, and edited the manuscript and have approved it for publication. Both 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.


Mitral valve chordae tendineae forces are elevated in the setting of mitral regurgitation (MR). Ring annuloplasty is an essential component of surgical repair for MR, but whether chordal forces are reduced after mitral annuloplasty has never been validated in vivo. Here, we present an extremely rare ovine case of natural, severe chronic functional MR, in which we used force-sensing fiber Bragg grating neochordae to directly measure chordal forces in the baseline setting of severe MR, as well as after successful mitral ring annuloplasty repair. Overall, our report is the first to confirm in vivo that mitral ring annuloplasty reduces elevated chordae tendineae forces associated with chronic functional MR. Ring annuloplasty is an essential component of mitral valve repair for the correction of mitral regurgitation (MR). Previous ex vivo studies have demonstrated that chordae tendineae forces are significantly elevated under simulated conditions of functional MR due to annular dilation and loss of leaflet coaptation  and thatA male Dorset sheep  was diagnosed on cardiac magnetic resonance imaging (MRI) with left atrial (LA), left ventricular (LV), and mitral annular dilation , as wellThe sheep was intubated, and a right thoracotomy was performed. A large 1.5 cm patent ductus arteriosus (PDA) was discovered, suggesting over-circulation as the etiology of LV dilation and functional MR. Retrospectively, the PDA could be identified on MRI, although dedicated sequences were not obtained B. The PDOne secondary chord to the anterior leaflet (AS) and two primary chordae, one each to the anterior (AP) and posterior leaflets (PP), were instrumented with custom-designed force-sensing fiber Bragg grating neochordae (FBGN) . For eacNext, the heart was re-arrested on CPB and the mitral valve was repaired with a 28 mm Carpentier–Edwards Physio II ring  F. Cross-Our report is the first to validate in vivo that mitral ring annuloplasty reduces elevated chordal forces associated with chronic functional MR. The in vivo forces we measured pre- and post-repair agreed well with previous ex vivo data assessing the impact of progressive annular dilation on chordal biomechanics .Our unique ovine case involved severe chronic MR that developed naturally. Chronic left-to-right shunting through a large PDA resulted in persistent left heart volume overload, leading to gradual LA and LV dilation  and ultiAlthough we draw inferences from only one animal, due to its extreme rarity, ex vivo experiments are currently unable to precisely replicate the motion of the mitral annulus, papillary muscles, and left ventricle wall, all of which directly influence valvular biomechanics. Furthermore, chordal forces cannot be safely analyzed in vivo in human patients with mitral valve disease, and no reproducible in vivo large animal model of natural MR exists. Indeed, to our knowledge, a PDA has never previously been reported to result in severe MR in a sheep . AlthougThe use of FBGN in an in vivo setting carries some inherent limitations. For example, it is possible that, in the course of replacing the native chordae with FBGN sensors, we may have inadvertently altered the MR disease state from the baseline. However, TEE imaging provided quantitative evidence of persistently severe central MR after FBGN implantation. In addition, the forces we measured in the in vivo state of severe, chronic functional MR were relatively similar to those observed in a previous ex vivo MR model . FurtherOverall, we report the first in vivo biomechanical validation that mitral ring annuloplasty reduces abnormally elevated chordae tendineae forces associated with chronic functional MR. Future long-term studies including sheep with induced MR versus healthy controls are required to confirm our observations.


In this editorial, we briefly summarize the International Conference on Intelligent Biology and Medicine 2019 (ICIBM 2019) that was held on June 9–11, 2019 at Columbus, Ohio, USA. Then, we introduce the two research articles included in this supplement issue. These two research articles were selected after careful review of 105 articles that were submitted to the conference, and cover topics on deep learning for drug-target interaction prediction and data mining and visualization of high-order drug-drug interactions. The International Conference on Intelligent Biology and Medicine 2019 (ICIBM 2019) had four keynote lectures, four eminent scholar talks, five tutorials and workshops, twelve concurrent regular scientific sessions, and one poster session. A total of 164 researchers attended the conference of which 79 were faculty/staff and 84 were trainees. The accepted papers and posters cover a wide range of topics in bioinformatics, next-generation sequencing, single cell analyses, systems biology, intelligent computing, data science, medical informatics and decision making, and others. Among 105 original manuscript submissions, two research articles are of special interest to drug-target interaction and drug-drug interaction prediction. The proposed medical informatics approaches are innovative and have strong translational potentials to facilitate decision making in clinical service. These two articles have gone through peer-review and revision before their final acceptance to this ICIBM2019 supplement issue.A key component in drug discovery is to identify protein targets and compounds that are active to the targets. Computational methods play an important role in predicting drug-target interactions (DTIs) due to its high throughput compared to the traditional experimental methods. Wang et al.  develop Adverse drug events (ADEs) often occur as a result of drug-drug interactions (DDIs). The use of data mining for detecting effects of drug combinations on ADE has attracted growing attention and interest. However, the existing approaches become inefficient from both computational and illustrative perspectives when considering more than three drugs in interactions. Yao et al.  develop Drug-target interactions and drug-drug interactions provide important medical information that can facilitate decision making in clinical settings when drugs are prescribed. Such research topics emerge at ICIBM 2019 and have drawn signification attention from the conference participants. New methods have been under development particularly via deep learning, large-scale data mining and visualization. We envision a growing interest of medical informatics approaches to address the related prediction problems. We also hope to organize more events similar to ICIBM 2019 to promote computational pharmacology in the areas of medical informatics and machine learning.


Toxicity studies for conventional oral drug formulations are standardized and well documented, as required by the guidelines of administrative agencies such as the US Food & Drug Administration (FDA), the European Medicines Agency (EMA) or European Medicines Evaluation Agency (EMEA), and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). Researchers tend to extrapolate these standardized protocols to evaluate nanoformulations (NFs) because standard nanotoxicity protocols are still lacking in nonclinical studies for testing orally delivered NFs. However, such strategies have generated many inconsistent results because they do not account for the specific physicochemical properties of nanomedicines. Due to their tiny size, accumulated surface charge and tension, sizeable surface-area-to-volume ratio, and high chemical/structural complexity, orally delivered NFs may generate severe topical toxicities to the gastrointestinal tract and metabolic organs, including the liver and kidney. Such toxicities involve immune responses that reflect different mechanisms than those triggered by conventional formulations. Herein, we briefly analyze the potential oral toxicity mechanisms of NFs and describe recently reported in vitro and in vivo models that attempt to address the specific oral toxicity of nanomedicines. We also discuss approaches that may be used to develop nontoxic NFs for oral drug delivery. Oral administration is one of the most popular routes for delivering drug formulations due to its convenience of dosing and excellent patient compliance. While conventional oral formulations are constructed to increase the drug’s systemic bioavailability, nanoformulations (NFs) are often fabricated to enable the precise release of the encapsulated drug to the disease site by active targeting -responsive targeting) and/or passive targeting (such as through the enhanced permeability and retention (EPR) effect) approaches ,2,3,4. TVarious materials, including inorganic  and organic  nanomaterials, have been used to fabricate oral NFs ,9,10,11.2) were found to suppress ROS toxicity, suggesting that ROS generation cannot be predicted solely from the size of NFs  thymidine into new strands of chromosomal DNA during mitotic cell division [3H] thymidine to cells undergoing DNA synthesis.The traditional method of identifying cell proliferation is by microscopically observing and counting cells. By such a method, mitotic progression can be observed in the presence of experimental NFs. The result is shown as the “mitotic index”, which represents the number of mitotic cells divided by the total number of observed cells . Measuredivision . Howeverdivision ,281. Brd2, then the single-stranded DNA of condensed chromatin from denatured nuclei is targeted using Apostain antibodies.Toxicity-induced DNA fragmentation and chromatin condensation can be captured by the terminal dUTP-transferase-mediated nick end labeling (TUNEL) assay  or stainThe antigen, Ki67, is a nuclear protein that is present during the active phases of the cell cycle  but absent from the resting state (G0 phase). It can be used as a cell proliferation marker, as a significant increase of Ki67 protein (pKi67) indicates active cell proliferation ,286. pKiGene expression changes have often been assessed by traditional methods, such as Northern blot analysis or ribonuclease protection assays (RPAs) ,290. NorAnother widely used method is real-time polymerase chain reaction (RT-PCR), which is a quantitative method for determining the number of copies of PCR templates like DNA or cDNA . RT-PCR Microarray analysis is a high-throughput method that can be used to measure the mRNA levels of a cell. It employs microchips coated with arrays of short DNA probes and can be used for large-scale screening . SamplesGuanine has the lowest reduction potential among the four DNA bases and is the most readily oxidized base . Under o3O4) NPs, were found to quench the fluorescence of the probe H2DCFDA [Most of the standardized toxicity assays were developed for evaluating conventional drugs or formulations ,296. Alt H2DCFDA ,300, cau H2DCFDA . Similar H2DCFDA . Sometim H2DCFDA , whereas H2DCFDA . For theThe toxicity of NFs can be built up in many ways; the general rule for avoiding nanotoxicity is to reduce the drug dosage by using targeted NP drug delivery systems and make major efforts to avoid using nondegradable NFs . Along wSome formulation strategies have shown promising results in their discovery stages. One such strategy is the use of mammalian cell-secreted exosomes ,77,308 A. For exIn cases where the use of metal, inorganic, carbon nanotube, graphene, and other exogenously sourced NFs cannot be avoided, surface modifications are necessary to prevent direct contact at the nano–bio interface ,315,316.Unlike other delivery routes, oral administration of NFs increases their exposure to widely diversified biological and chemical conditions, including enzymatic- and microbiota-related digestion and ionic strength and pH variations. The harsh conditions of the GI tract challenge the stability of NFs. Nanotoxicity typically originates from the instability of NFs and their unique physicochemical properties. The aggregation and dissociation of NFs in luminal fluids and at the mucus–epithelial cell interface also affect their topical toxicology behaviors.Researchers urgently need to establish a combination of complementary models for assessing oral nanotoxicity. Although in vivo and ex vivo models can provide useful toxicity information related to human cells and tissues, these models cannot offer accurate information on the absorption, distribution, metabolism, and excretion (ADME) fate of NFs, nor can they provide drug metabolism and pharmacokinetics (DMPK) profiles of the NFs in the host. At present, we still require in vivo models, despite the booming of new animal-replacing organ-on-a-chip and microfluidic technologies.Enormous efforts have been made to establish detection methods for evaluating the safety of emerging nanomaterials. Many of these methods are derived from the protocols for assessing conventional formulations and thus are not explicitly designed for evaluating nanotoxicity. Given that NFs interfere with optical-based detections in many ways, unmodified methods will undoubtedly lead to inconsistent results. Instead, NFs should be removed before samples are processed for optical-based quantitation, with the goal of minimizing such interference. An obvious yet underappreciated issue is that most of the applied methods lack a stringent method validation step. This is a normal phenomenon in the biomedical field, as many biomedical researchers are more familiar with ready-to-use protocols. Validating a newly established method tends to fall more within the research interests of analytical chemists or analytical biochemists.Nanotoxicology is still a developing discipline. New technique-based platforms (such as mass-spectrometry-based proteomics and microarray-based transcriptomics) are powerful tools for investigating toxic responses of the cells or tissues at a global level, providing comprehensive information that cannot be obtained with traditional probe- and antibody-based assays. These platforms can also largely bypass the NFs’ optical interference issues: the hyphenated detection techniques, such as LC-MS or capillary electrophoresis–mass spectrometry (CE-MS), are equipped with strong separation capabilities and are built for multi-omics studies that tackle samples within a complex biomatrix. Going forward, nanotoxicology studies can make full use of these new techniques, which will open our minds to designing better toxicology models for studying nanotoxicity.