Objective: Due to an ever-increasing ageing population and limited available data around the use of thoracic endovascular aortic repair (TEVAR) in elderly patients, investigating its efficacy and safety in this age cohort is of vital importance. We thus reviewed the existing literature on this topic to assess the feasibility of TEVAR in elderly patients with severe thoracic aortic pathologies. Methods: We identified all original research studies that assessed TEVAR in elderly patients published up to 2023. Morbidity, as assessed by neurological and respiratory complications, endoleaks, and length of stay, was the primary endpoint. Short-term mortality and long-term survival were the secondary endpoints. The Mantel–Haenszel random and fixed effects methods were used to calculate the odds ratios for each outcome. Further sensitivity and subgroup analyses were performed to validate the outcomes. Results: Twelve original studies that evaluated elective TEVAR outcomes in elderly patients were identified. Seven studies directly compared the use of TEVAR between an older and a younger patient group. Apart from a shorter hospital stay in older patients, no statistically significant difference between the morbidity outcomes of the two different cohorts was found. Short-term mortality and long-term survival results favoured the younger population. Conclusions: The present meta-analysis indicates that, due to a safe perioperative morbidity profile, TEVAR should not be contraindicated in patients based purely on old age. Further research using large patient registries to validate our findings in elderly patients with specific aortic pathologies and both elective and emergency procedures is necessary. Thoracic endovascular aortic repair (TEVAR) has achieved rapid adoption as a treatment modality for thoracic aortic disease since its introduction in 1992. The minimally invasive nature of TEVAR and its short-term safety and efficacy have led to its expanding utilization as the primary treatment approach for various aortic pathologies, including penetrating atherosclerotic ulcers and aortic transections .While there is substantial evidence supporting the use of TEVAR in nonelderly patients with a low-risk factor profile, concerns have been raised about its suboptimal outcomes in certain populations, particularly the elderly. Multiple studies have shown increased postoperative morbidity, such as stroke and pulmonary complications ,5,6, andThus, the question of whether TEVAR should be offered to this age group remains unanswered. This is particularly important as, due to an ever-increasing ageing population, it is projected that by 2050 more than 50% of aortic dissections will occur in patients over 75 years old ,9. In liIn this context, we designed a systematic review and meta-analysis aiming to evaluate TEVAR morbidity and mortality outcomes in elderly patients. This was achieved by comparing the morbidity and mortality outcomes of TEVAR between elderly and nonelderly patients. The primary endpoint was perioperative morbidity defined as (1) endoleaks, (2) neurological complications, (3) respiratory complications, and (4) length of hospital stay (LoS), and secondary outcomes included in-hospital mortality and long-term survival following TEVAR.This study was conducted in accordance with a protocol agreed upon by all authors and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines . The MedData extraction was performed independently by two reviewers (AF and SB) into a comprehensive Excel spreadsheet. In cases of discrepancy, studies were discussed in group meetings with the senior authors (DEM and TA) until a consensus was reached. The following information was extracted from each study: first author, year of publication, study-population characteristics, study design, baseline patient characteristics, number of patients operated using TEVAR, the age limit for defining the elderly population, and postoperative outcomes.This study attempted to compare the outcomes of elective TEVAR in elderly and nonelderly patients. Studies which reported on outcomes of urgent TEVAR only and on debranching TEVAR were excluded. The majority of the studies included, however, did not distinguish or conduct subgroup analysis between elective and emergency TEVAR. The elderly patients were defined as the experimental group and nonelderly patients as the control group. From the included studies, eight studies used 80 years of age for defining elderly ,13,14,15The baseline preoperative patient characteristics of the included studies are presented in Seven studies directly compared TEVAR outcomes between elderly and nonelderly patients ,12,14,17The primary endpoint of this study was to assess and compare the perioperative morbidity of TEVAR between elderly and nonelderly patients. As mortality trends often favour younger populations, the decision to select morbidity outcomes as the primary endpoint was made. Morbidity was defined based on the following outcomes: (1) neurological complications (2), endoleaks, (3) respiratory complications, and (4) length of hospital stay. Due to the heterogeneity of definitions of neurological complications provided by the papers examined, the umbrella term ‘neurological complications’ was tilized, which involved cerebrovascular accidents (CVAs) and paralysis/paraplegia. Studies that reported separately on the aforementioned outcomes were combined and reported under ‘neurological complications’.The secondary endpoint of this study was mortality, defined as a (1) short-term mortality, specifically either 30-day or in-hospital mortality, and (2) overall survival following TEVAR procedures. Both 30-day mortality/in-hospital mortality and overall survival were compared in elderly and nonelderly patients.Study quality and risk of bias assessment were performed using the Risk of Bias in Non-Randomized Studies of Interventions tool (ROBINS-I) to evaluate non-RCTs . No RCTsWhere available, data on the outcomes of emergency and elective TEVAR were used for a sensitivity analysis to explore how the outcomes in the elderly population were affected by the nature of the operation. The only outcome in which there was sufficient data to enable such a subgroup analysis regarding emergency and elective operations was short-term mortality.The Mantel–Haenszel random and fixed effects methods were used to combine the odds ratio (OR) for outcomes of interest allowing for comparison between elderly and nonelderly populations. Due to the heterogeneity of the studies’ populations and surgical techniques, a random effect analysis was necessary to account for the variance within each study and between them. For funnel plots, the fixed-effect method was chosen because their purpose is not to estimate an overall effect size but rather to visually inspect the distribution of study results and identify potential bias . Two strp-value of less than 0.05. Data and statistical analysis were performed using RevMan V5.4 (The Cochrane Collaboration) and IBM SPSS Statistics .Pooled overall survival analysis was performed using the published Kaplan–Meier graphs from the included studies using a two-stage approach . In stagThe total number of studies included in this analysis was 12 . All theFive included studies investigated the number of neurological complications between the elderly and nonelderly populations. Thus, a total of 1186 elderly and 5618 nonelderly patients were compared for this outcome. Endoleaks in both populations were reported in three of the included studies, amounting to a total number of 118 elderly and 399 younger patients. As shown in Data on respiratory complications among both age cohorts were documented in four studies. This led to a comparison of 1128 elderly and 5313 nonelderly patients. There was no significant difference in the incidence of respiratory complications  a.The duration of hospital stay in both age groups was reported in four studies. Hence, 757 elderly patients and 3906 nonelderly were pooled for this outcome. As this endpoint was documented in seven studies for both cohorts, a comparison of short-term mortality was carried out between 1288 elderly patients and 5925 younger patients. The number of studies reporting on elective versus emergency TEVAR procedures in the elderly was three. This translated to a total of 152 patients undergoing elective operations and 47 patients undergoing emergency TEVAR. A comparison regarding short-term mortality between elective and emergency TEVAR procedures in elderly patients revealed a significantly higher mortality in emergency repairs  a.n= 633) and nonelderly (n = 1897) and Kaplan–Meier curves were constructed to analyse overall survival. Patients in the elderly group had a mean OS of 42.9 months compared to 50.6 months for nonelderly patients .Funnel plots investigating publication bias for each outcome are presented in No significant difference was found in the leave-one-out method when comparing elderly vs. nonelderly patients in the following primary endpoints: 30-day mortality, neurological complications, and respiratory complications. A significant difference was observed when excluding the studies by Kpodonou, Alnahhal, and Kern et al. ,14,17 reThis systematic review and meta-analysis demonstrated that the morbidity associated with TEVAR did not differ significantly between elderly and nonelderly patients. A shorter length of hospital stay was also identified in the older age group. Regarding our secondary endpoints, short-term mortality was found to be lower in the nonelderly population. In addition, younger patients also exhibited higher long-term overall survival rates with lower long-term mortality rates.Neurological complications often have significant long-term health implications for patients, especially for elderly ones, and are commonly reported by studies ,22. Our Endoleaks represent a common reason for reintervention following endovascular aortic repair, contributing to significant postoperative morbidity ,24. No sRespiratory complications and postoperative length of hospital stay in the elderly after TEVAR are less reported in the literature. In a large US nationwide study, TEVAR was associated with decreased respiratory complications and LoS compared to open aortic repair, even though the TEVAR population was significantly older . Hence, n = 7). Previous studies have also demonstrated that the short-term mortality of elderly patients post-TEVAR is higher compared to the nonelderly. Using data from large multinational registries, Hellgren et al. identified a higher short-term death rate in patients over 80 years old undertaking TEVAR for a variety of aortic pathologies [Short-term mortality, either reported as 30-day or in-hospital mortality, was the endpoint most commonly documented by studies comparing post-TEVAR outcomes between elderly and younger patients (hologies . Apart fhologies . Elderlyhologies . HoweverAs the incidence of aortic pathologies in elderly patients continues to rise ,7 and thThe population included in this review consisted of patients undergoing TEVAR for a variety of aortic pathologies. As most studies did not report separate outcome results for each diagnosis; a comprehensive analysis of how post-TEVAR morbidity and mortality varied with different aortic presentations could not be conducted. Comparisons of patient outcomes undergoing TEVAR for aortic aneurysms and dissections have yielded variable results. Although some studies report similar postoperative morbidity and mortality ,36, infeThe limitations of this meta-analysis greatly depend on the individual studies used, which were retrospective cohort studies, as no relevant RCTs were identified during the literature search. Due to the nature of the comparison, an RCT between the two age groups might not be feasible. Hence, future research using large TEVAR registries should be prioritised. Until then, this systematic review and meta-analysis remains the best level of evidence around TEVAR use in the elderly. In addition, as mentioned, renal complications and the effect of different aortic conditions, and the urgency of the procedure on outcomes after TEVAR, were not explored. There was also a lack of available information about other important procedural details, such as renal complications, type of anaesthesia, and duration of the TEVAR procedure. We strongly recommend that future studies report on these variables, as they can influence surgical outcomes. Patient data were derived from Kaplan–Meier curves, and not individual patient information, thus further limiting us from conducting even more subgroup analyses based on various patient characteristics. Different selection criteria between studies and confounding variables are also significant factors that could not be controlled.The overall findings of this systematic review and meta-analysis suggested that morbidity following TEVAR, assessed by the incidence of neurological complications, respiratory complications, and endoleaks, was similar in elderly and nonelderly patients. Short-term mortality and long-term overall survival post-TEVAR favoured, as expected, the younger population. Due to its safe perioperative morbidity profile, it can be concluded that patients should not be excluded from elective TEVAR interventions purely based on age criteria.


Autoimmune blistering diseases (AIBD) comprise a heterogeneous group of uncommon disorders of the skin and mucous membranes, characterised by antibodies targeting structural proteins within epithelial tissue and the underlying basement membrane. There can be significant overlap in clinical presentation of these diseases and accurate diagnosis relies on the detection and characterisation of relevant autoantibodies. Immunofluorescence provides the gold-standard diagnostic tool for these diseases, identifying both tissue-bound autoantibodies in biopsy material using direct immunofluorescence and circulating antibodies in serum through indirect immunofluorescence. Following advances in the identification and subsequent characterisation of numerous antigenic targets in these diseases, the development of antigen-specific tests, in particular, enzyme-linked immunosorbent assays on serum specimens, has provided a third key tool to not only identify, but also quantify AIBD autoantibodies. This quantification has proven particularly useful in monitoring disease activity and informing clinical management decisions. Accurate diagnosis of these diseases is important since optimal treatment strategies differ between them and, prognostically, some diagnoses are associated with an increased risk of malignancy. This review outlines the molecular pathology underlying the major AIBD and describes how the three principal techniques can be used in combination, to provide best practice for diagnosis and treatment monitoring. Autoimmune blistering diseases (AIBD) encompass a group of serious disorders of the skin and mucous membranes which are characterised by the production of autoantibodies which typically target structural proteins within epithelial tissue and in the underlying basement membrane zone, connecting epithelium with either dermis in skin or the lamina propria in mucosal tissue. This results in loss of structural integrity of these tissues and consequent blistering . Prior tAIBD can be divided into two categories; those producing autoantibodies targeting antigens within the epithelium, termed the intra-epithelial autoimmune bullous diseases and those which generate autoantibodies that bind to a distinct group of structural proteins found in the region beneath the epithelium, comprising the sub-epithelial autoimmune bullous diseases . More spvice versa [To maintain structural integrity, keratinocytes, the predominant epithelial cell type within human skin and mucosae, anchor themselves to adjacent keratinocytes via desmosomes, a type of cell junction. Desmosomes facilitate the direct connection of cytoskeletal keratin filaments between cells and are randomly located at the plasma membrane . They arce versa . In humace versa . All mayThe interface where epithelial cells attach to the underlying connective tissue is termed the basement membrane zone (BMZ) and is sometimes used synonymously with the dermo-epidermal junction in skin. It is a distinct structure, comprising four major ultrastructural regions and describes the area where focal attachment structures within basal keratinocytes interact with a series of molecules in the acellular space directly beneath the epithelium, to form a tight connection between the two layers, which also creates a selective barrier .Within basal keratinocyte plasma membranes, structures called hemidesmosomes serve to bundle keratin intermediate filaments of the cytoskeleton together and attach them to other members of the plakin superfamily, in particular, plectin and dystonin (commonly referred to as BP230 or bullous pemphigoid antigen 1/BPAG1) . These pThe sublamina densa region (sometimes termed the fibrillar zone), directly beneath the lamina densa, comprises collagen VII anchoring fibrils, anchoring plaques composed of collagen IV and laminin, and collagen and elastic fibres. The anchoring fibrils not only link the lamina densa with the anchoring plaques of the underlying papillary dermis  but also interact with laminin 332 in the lamina lucida, thus providing a direct link between hemidesmosomes in basal keratinocytes and papillary dermis/lamina propria, which is pivotal in maintaining a strong attachment between the epithelium and underlying connective tissue .Autoantibodies directed against epitopes on the transmembrane adhesion molecules of desmosomes have been shown to be pathogenic in the intra-epithelial blistering diseases which comprise the pemphigus family , 18, allAntibodies of the IgG isotype predominate in most cases of pemphigus. However, a small proportion of pemphigus patients produce IgA autoantibodies exclusively, which are most commonly directed against the desmoglein homolog, desmocollin-1 .Prior to the advent of corticosteroids  in the 1950s, approximately 75% of pemphigus cases were fatal . HoweverIn contrast to the pemphigus family, individuals with diseases in the sub-epithelial blistering disease group produce autoantibodies which target proteins associated with the basement membrane zone, producing tense, pruritic blisters, clinically, which are sub-epithelial on histological examination . The mosMucous membrane pemphigoid (MMP) is distinguished from BP, clinically, by predominant mucosal involvement with oral and ocular sites being the two most commonly affected areas . In addiLinear IgA disease (LAD) is characterised, immunopathologically, by production of BMZ-localising IgA antibodies, in which the soluble, ectodomain of BP180, rather than the (uncleaved) NC16A region, is the principal autoantigen . ConversThe most recently identified pemphigoid variant is characterised by the presence of antibodies against the laminin-γ1 chain, which was shown to be the target antigen in what had previously been termed anti-p200 pemphigoid , althougEpidermolysis bullosa acquisita (EBA) is a sub-epithelial blistering disease that can be difficult to distinguish from BP, clinically, but can be diagnosed by detection of autoantibodies binding to type VII collagen .Dermatitis herpetiformis (DH) is typically seen as the cutaneous manifestation of coeliac disease and most commonly presents as pruritic, symmetrically distributed papules and blisters at the extensor surfaces of upper and lower limbs . DH is ain situ tissue autoantibodies, indirect immunofluorescence microscopy, using patient sera and a range of tissue substrates to detect and titrate circulating autoantibodies and, less commonly, ELISA of serum specimens, to detect and quantitatively determine levels of autoantibodies to defined autoantigens in these diseases.There are currently three widely used techniques to assist clinicians in the diagnosis and monitoring of AIBD: direct immunofluorescence microscopy of biopsy material, for detection of in situ and other proteins of relevance to AIBD. A primary antibody directed against the protein of interest and conjugated with a fluorescent dye  is added to tissue sections derived from a patient biopsy. Any specific immune complexes formed during incubation of the tissue sections with these antibodies are then visualised using fluorescence microscopy with appropriate filters. The most commonly used panel of fluorescently conjugated antibodies detects tissue-bound IgA, IgG, and IgM immunoglobulins, in addition to the C3c subunit of complement and fibrinogen [DIF is a single-step procedure used to identify antibodies bound to cutaneous or mucosal antigens brinogen . The senbrinogen . HoweverBiopsy material for direct immunofluorescence cannot be fixed in formalin, prior to analysis, since the resultant cross-linking of proteins inhibits antibody binding and even short exposure of a few minutes can render specimens unsuitable for DIF analysis . The traIIF is a two-step procedure used to identify circulating autoantibodies to cutaneous or mucosal antigens in patient serum. These antibodies are most commonly of IgG and IgA isotypes. Although serological antibody concentrations are typically much lower than those found in tissues of patients with AIBD and DIF has a higher sensitivity for detection of these diseases , IIF is Following blood sample collection, serum is separated by centrifugation and can be stored at 4°C for up to 1 month, prior to analysis. To detect circulating autoantibodies, tissues containing corresponding antigens must be used as a substrate. Two principal tissue types are commonly employed. Monkey oesophagus provides a rich source of desmoglein proteins, particularly desmoglein-3 and is therefore the substrate of choice for the detection of pemphigus vulgaris antibodies . CommercIn addition to normal human skin substrate, use of human salt-split skin provides a valuable third substrate for IIF. The split-skin method is a relatively straightforward and reliable technique for distinguishing between epidermal and dermal-binding autoantibodies in sub-epithelial AIBD . It reliA fourth, less frequently used IIF substrate is rat (or monkey) urinary bladder, or similar tissues, that contain transitional epithelium. Unlike stratified squamous epithelium found in skin, transitional epithelium does not produce DSG1 or DSG3, but expresses significant levels of desmosomal plakin family proteins, especially envoplakin, periplakin and desmoplakin . AntibodTo detect circulating autoantibodies in a patient with a suspected AIBD, slides prepared with 4–5 μm frozen sections of appropriate tissue substrates are incubated with diluted patient serum. After incubation, slides are washed to remove unbound antibodies and incubated again with fluorescently labelled anti-human IgG or IgA conjugates. They are then washed again and dried, prior to mounting and subsequent fluorescence microscopy. Results are typically reported either qualitatively (positive or negative for intercellular or basement membrane zone fluorescence) or semi-quantitatively  for both IgG and IgA conjugates . As withIn addition to IIF techniques, circulating antibodies in serum from patients with AIBD can be detected using ELISA. Unlike IIF, ELISA techniques are specific for known autoantigens and offer higher sensitivity and a greater degree of quantitation , which cELISAs are typically performed using commercial kits to assess the most commonly occurring autoantibodies seen in AIBD, i.e., DSG1 & DSG3 antibodies in pemphigus , BP180 &Application of DIF methodologies to tissue biopsies and IIF protocols to serum specimens from patients with suspected AIBD produces two principal types of fluorescence pattern. Those patients with intra-epithelial AIBD display an intercellular pattern, as a result of antibody binding to disrupted desmosomes located on epithelial cell membranes, whereas patients with sub-epithelial AIBD exhibit sharp, linear fluorescence at the basement membrane zone, due to antibody binding to hemidesmosomal targets in these tissues. Further diagnostic clues in DIF are provided by spatial localisation of fluorescence and the combination and relative fluorescence intensity of positive conjugates observed. A combination of substrate positivity, fluorescence pattern seen (intercellular vs. linear), conjugate positivity (IgG vs. IgA), split skin analysis and relevant ELISA positivity can be used to provide a diagnosis from a serum sample . SpecifiDirect immunofluorescence in patients with all types of pemphigus produces a characteristic, sharp, “chicken-wire” pattern with IgG and/or C3 conjugates, localising to epithelial cell membranes . IgA mayMonkey oesophagus is most commonly used as a substrate to titrate IgG antibodies from the serum of patients with PV, whereas patients with PF exhibit IgG antibodies which bind preferentially to human skin substrate. This substrate distinction is not absolute, since serum from patients with the mucocutaneous variant of PV, who produce autoantibodies against both DSG1 and DSG3, display immunofluorescence on both substrates and monkey oesophagus typically also fluoresces in PF. Quantitation of specific anti-DSG1 and DSG3 antibodies can be achieved with DSG ELISA and facilitates pemphigus sub-typing. Patients with DSG1 antibodies alone are defined as having PF, whereas those with anti DSG3  are predominantly patients with PV . DSG antIIF using rat transitional epithelium substrate can be performed on sera from patients with suspected PNP, who typically also express DSG3 ± DSG1 antibodies. PNP can be differentiated from PV by the additional finding of intercellular IgG deposition on transitional epithelium , due to Diseases of the pemphigoid sub-family show a linear deposition pattern of IgG and/or C3 at the BMZ of epithelial tissue by DIF, due to the hemidesmosomal location of antigens targeted in these diseases . IgA linIndirect immunofluorescence on sera from BP patients, using split normal human skin as a substrate, typically produces circulating autoantibody titres that are higher than those seen in pemphigus. In addition, IIF permits differentiation of this disease from EBA, in cases where biopsy material is not available for DIF analysis, in addition to identification of patients with a heterogeneous autoantibody response, which may be observed as immunolocalisation to both the roof and base on the split skin substrate. A specific ELISA for anti BP180 and/or BP230 antibodies provides confirmation of a BP diagnosis with approximately 90% sensitivity . BP180 EPatients with linear IgA disease show a bright, linear deposition of IgA at the BMZ on DIF. In addition, there may be a weak linear IgG and/or C3 band present in some patients. The relative intensities of the IgA and IgG deposits can be useful in differentiating linear IgA disease from MMP, if this is unclear from the clinical or histological presentations. Since BP180 is the predominant target antigen in linear IgA disease, split-skin IIF in these patients typically shows localisation of IgA to the roof of the split, although titres are lower, compared with IgG levels seen in pemphigoid patients. A small proportion of linear IgA cases show base-binding localisation, which may be attributable to collagen VII reactivity, since this is the predominant antigen in cases of drug-induced linear IgA disease precipitated by the antibiotic, vancomycin . CommercDIF on biopsies from EBA patients shows linear deposition of IgG, with or without C3, at the basement membrane zone, in a similar pattern to that seen in BP patients. However, careful microscopic examination may indicate a slightly thicker band than that seen in BP, with a u-serrated pattern seen at higher magnifications . Salt-spBiopsies from patients with DH exhibit a characteristic pattern of granular (sometimes fibrillar) deposition of IgA at or just below the dermo-epidermal junction . DepositDirect immunofluorescence remains the gold standard for the diagnosis of AIBD from cutaneous and mucosal biopsies. Advances in the characterisation of the autoantibodies produced in these diseases and their antigenic targets has facilitated the development of additional serological assays which can be used both to confirm and refine the diagnosis indicated by DIF. In particular, the availability of autoantibody-specific ELISA has increased serological sensitivity of AIBD detection over the last 25 years and provided quantitation of antibody production that has become a valuable tool in the therapeutic management of patients. Combined use of DIF, multi-substrate IIF and ELISA methodologies currently provides the optimal strategy for diagnosis and immunological monitoring in these diseases. Future developments will likely focus on the continued development of multiplex assays that facilitate simultaneous measurement of multiple antibodies in a single serum sample, including multi-substrate BIOCHIP mosaics for IIF , multipa


Dengue is the most common arboviral disease worldwide and the leading cause of febrile illness among US travelers returning from the Caribbean. Infectious disease surveillance of international travelers is an effective method for detecting pathogens circulating in resource-limited areas. Due to the large number of infected travelers diagnosed in Florida, we hypothesized that we could use travel surveillance data to reconstruct local outbreaks in the Caribbean, focusing on Cuba, Dominican Republic, Haiti, Jamaica, and Puerto Rico.We obtained reports of international travel-associated dengue virus infections diagnosed in Florida between 2009-2022 from the Florida Department of Health (FL DOH) mosquito-borne disease surveillance system. We calculated travel incidence rates using the yearly number of air travel passengers arriving in Florida from international destinations. We calculated local incidence rates from yearly dengue case counts from Caribbean countries and territories with local transmission, provided by the Pan-American Health Organization (PAHO).Most infected travelers recently arrived from Cuba; however, travel dengue incidence poorly correlated with local dengue incidence there (R = 0.502), raising concerns about the accuracy of local surveillance. Conversely, travel dengue incidence from the Dominican Republic, Jamaica and Puerto Rico strongly correlated with local incidence , implying that travel data is a good predictor of local outbreaks in these countries. We discovered that during outbreak years different dengue virus (DENV) serotypes were responsible for outbreaks on each island, demonstrating that regional outbreaks are likely due to overall favorable conditions, such as mosquito abundance, and not due to a new introduction or mutation.Travel surveillance data provides a good approximation of local dengue incidence in some Caribbean countries but does not correlate strongly with local case reporting from Cuba and Haiti. This raises the possibility of using real-time tracking and sequencing of dengue cases in Florida, paired with travel surveillance data, to detect Caribbean outbreaks as they are occurring to inform public health policy in resource-limited countries.All Authors: No reported disclosures


Altering the diet to treat disease dates to c. 400 BC when starvation was used to reduce seizures in persons with epilepsy. The current diversity of symptomology and mechanisms underlying autism spectrum disorders (ASDs) and a corresponding lack of disorder-specific effective treatments prompts an evaluation of diet as a therapeutic approach to improve symptoms of ASDs. In this review article, we summarize the main findings of nutritional studies in ASDs, with an emphasis on the most common monogenic cause of autism, Fragile X Syndrome (FXS), and the most studied dietary intervention, the ketogenic diet as well as other dietary interventions. We also discuss the gut microbiota in relation to pre- and probiotic therapies and provide insight into future directions that could aid in understanding the mechanism(s) underlying dietary efficacy. FMR1) gene located at Xq27:3 on the X-chromosome. An expansion greater than 200-CGG repeats leads to inactivation of the FMR1 gene and loss of its protein product fragile X messenger ribonucleoprotein (FMRP) (Fragile X Syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disability, autistic-like behaviors and seizures . Children (FMRP) . Lack ofn (FMRP) .FXS is the most prevalent genetic cause of autism spectrum disorders (ASDs) . The etiFmr1 gene (the homologous gene in mouse) can affect gut microbiota . Supplementation of macro or micronutrients that are deficient in ASDs, and (2). Avoiding nutrients and bioactive food components that exacerbate adverse phenotypes or cause illness. Among the micronutrients, different vitamins and minerals including fat-soluble vitamins , water-soluble vitamins , and minerals  are important for overall nutrition. In addition, dietary fatty acids, prebiotics, and probiotics have been used as supplements to treat ASDs . In contRecent studies indicate significant advances in the early detection of ASDs and preclinical research and clinical trials have begun to assess the efficacy of non-pharmacological therapies at earlier ages. Micronutrients may be an important component of ASD etiology, though many results are conflicting. For example, in a 2022 systematic review, authors describe that vitamin D can improve hyperactivity in ASD, but their research did not show enough beneficial evidence for improving ASD-related symptoms . In addiFmr1 knockout (KO) mice were supplemented with alpha-tocopherol, which resulted in a reduction of free radicals causing less oxidative stress along with cytokine production and reduced macroorchidism. The alpha-tocopherol treatment also improved behavioral abnormalities and reduced learning deficits in KOFmr1 mice  fatty acids (FA) are essential for normal growth and maintenance of body functions; therefore, many studies have been performed to test their efficacy in mitigation of ASD traits. Ω-3 fatty acids were administered to activity . FXS micactivity . Consideactivity , it is iThe classic ketogenic diet was introduced in 1921 to replace starvation in the treatment of epilepsy . The ketRecent studies in ASD rodent models indicate that the KD improves core behavioral symptoms albeit there were some sex and genotype-specific differences . PrelimiKetogenic diet vs. gluten free casein free diet in autistic children: a case-control study, Others have reviewed current nutritional approaches in managing ASDs, the fundamental metabolic processes that promote brain health, and case and clinical studies testing the KD in ASD . Here, wTherapeutic use of carbohydrate-restricted diets in an autistic child; a case report of clinical and 18FDG PET findings, In their paper published in Metabolic Brain Disease in 2018 and entitled, A modified ketogenic gluten-free diet with MCT improves behavior in children with autism spectrum disorder, published in Physiology and Behavior in 2018, In their paper entitled, Metabolic framework for the improvement of ASDs by a modified ketogenic diet: a pilot study, published in Journal of Proteome Research in 2020, In their paper entitled, It remains to be determined if the positive effects associated with the KD in humans can be maintained after discontinuation of the diet. In rats, after 1 week cessation of the KD, social activity deficits returned to control levels , but in KOFmr1 mice with a ketogenic diet attenuated seizures and altered diurnal activity levels with sex and age specific effects  have been used for the treatment of Crohn’s disease, celiac disease, ulcerative colitis, diverticulitis, and chronic diarrhea and are currently receiving attention for their potential in treating neurological disorders . The purKOFmr1 mice on single-source soy protein-based diets increased seizures and weight gain (5) / estrogen receptor dependent signaling, and/or activation of the immune system by soy bioactive components including phytoestrogens and agrochemicals  along with a prebiotic agent, Bovine Colostrum Product (BCP), improved overall aberrant behaviors and GI disturbances while decreasing inflammatory cytokines including IL-13 and TNF-alpha . The treated group exhibited significant improvements in social behaviors and experienced less abdominal pain in bowel movements indicating the positive influence of prebiotic treatment in ASDs ] treatment in young children with ASDs on an exclusion (gluten and casein) diet  were assessed on neuroinflammation and gut dysbiosis in an autism rodent model [proprionic acid (PPA) treated golden Syrian hamsters]. This study showed significant improvements in gut-microbial composition and neuroinflammation in PPA-treated hamsters . Interesin) diet .Streptococcus, Lactococcus, and Saccharomyces   . Childretipation . Conversith ASDs . These iLactobacillus plantarum WCFS1, for 12 weeks and showed improved GI pathologies and significant amelioration of ASD behaviors. This study also reported an increase in Lactobacillus and Enterococci with a decrease in Clostridium coccoides in children with ASD compared to the placebo group  showed reduced antisocial behaviors and alteration in the gut microbial population  in C57BL/6J pregnant mice every 24 h for up to 21 days along with prebiotics  improved ASD like characteristics in offspring . ABA is a strategy to understand and alter the behaviors of young children with ASDs. ABA itself is not a therapy but a combination of different strategies and techniques to help people learn new skills and behaviors. The ABA approach has been shown to reduce food refusal and food selectivity while increasing interest in novel food, ultimately promoting consumption of diverse nutrients in children with ASDs. In addition, social story interventions have gained attention among behavioral scientists to improve social skills in people with ASD. Social story intervention is a technique where stories are used to increase engagement in socially appropriate mealtime attitudes in children with ASDs . Others Progress in ASD research, particularly in FXS, has provided a better understanding of the core symptoms and identification of potential treatment options. However, differences in underlying mechanisms leading to convergent and divergent phenotypes between FXS and ASDs as well as lack of consistent results in both preclinical and clinical studies are important issues. In addition, the major focus of research in neurodevelopmental disorders is the brain with minimal efforts aimed at the other organ systems or a whole-body approach. A plethora of nutritional interventions have been assessed in both animal and human models without conclusive results. Generally, small sample sizes, lack of appropriate controls, ambiguous results, and lack of technical or economic support limit preclinical, clinical, and translational ASD nutrition studies. Moreover, translation of nutritional interventions from animals to humans yields inconsistent results. Animal testing designs may not always be applicable for human trials due to genetic and epigenetic factors, subject variation and altered metabolism between species.Fmr1KO mice identified the rodent diet in the Methods section of their papers . (2) Nutritional interventions should be tested in multiple rodent models of ASDs to assess the generalizability of the findings. There are a plethora of genetic mutations contributing to ASDs and it will be important to identify diet-induced effects as a function of specific autism mutations, behaviors and biomarkers to understand both complementary and divergent mechanisms. (3) The mechanism(s) underlying diet effects need to be determined, i.e., microbiota composition, gut permeability, immune system activation, epigenetics, mitochondrial function, etc. as a function of autism genetic mutations and diet. Of note, there is a growing body of knowledge regarding mitochondrial dysfunction in ASDs including FXS (We propose the following future directions to address rigor and reproducibility in and promotion of nutrition research related to ASDs: (1) Publications must report details on rodent diet. Only 13% of publications reporting behavior results in ding FXS . And (4)ding FXS . There iAll authors contributed to the writing and editing of the manuscript.


Inflammation impacts human hematopoiesis across physiologic and pathologic conditions, as signals derived from the bone marrow microenvironment, such as pro-inflammatory cytokines and chemokines, have been shown to alter hematopoietic stem cell (HSCs) homeostasis. Dysregulated inflammation can skew HSC fate-related decisions, leading to aberrant hematopoiesis and potentially contributing to the pathogenesis of hematological disorders such as myelodysplastic syndromes (MDS). Recently, emerging studies have used single-cell sequencing and muti-omic approaches to investigate HSC cellular heterogeneity and gene expression in normal hematopoiesis as well as in myeloid malignancies. This review summarizes recent reports mechanistically dissecting the role of inflammatory signaling and innate immune response activation due to MDS progression. Furthermore, we highlight the growing importance of using multi-omic techniques, such as single-cell profiling and deconvolution methods, to unravel MDSs’ heterogeneity. These approaches have provided valuable insights into the patterns of clonal evolution that drive MDS progression and have elucidated the impact of inflammation on the composition of the bone marrow immune microenvironment in MDS. Inflammation is a complex biological response that occurs in the body when it is exposed to various triggers, such as an infection, tissue damage, or autoimmune disorders . While iHematopoietic stem cells (HSCs) are the foundation of the hematopoietic system that are responsible for the continuous production of all blood cell lineages throughout life. HSCs possess the unique ability to self-renew, generating identical copies of themselves, and differentiating into different cell types, including red blood cells, white blood cells, and platelets . InflammThe involvement of inflammation and innate immune responses in MDS pathogenesis have been recently reviewed elsewhere and are beyond the scope of this review ,5,6,7,9.Here, we aim to highlight recent significant advancements in understanding the mechanisms by which innate immune signaling and inflammation contribute to the development of MDS. A common theme that has emerged from recent studies is the induction of HSC expansion and altered responses in MDS HSC due to inflammation. This contributes to sustained myelopoiesis and a more competitive advantage compared to that of normal cells. Furthermore, we place special emphasis on the increasing importance of employing multi-omic approaches to comprehend immune dysregulation in MDS, with a focus on the role of single-cell profiling and deconvolution methods.Inflammation impacts human hematopoiesis across physiologic and pathologic conditions, such as infections, autoimmune disorders, and ageing. It is widely recognized that the activation of the immune system due to an infection or inflammation results in the activation of normal HSCs, ultimately causing to loss of their normal self-renewal capability . In respMDSs encompass a group of BM failure disorders distinguished by the presence of myeloid cell dysplasia. This is a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis, resulting in the dysplastic and insufficient production of blood cells. In addition to inefficient hematopoiesis, a component of chronic inflammation has been consistently reported. Although the exact etiology of MDS remains elusive, emerging evidence suggests the significant involvement of innate immune signaling and inflammation in its pathogenesis ,6,7. AltasDnmt3a, Tet2, and Asxl1) are also often found in other myeloid malignancies, such as MDS [Since MDSs primarily affect older individuals, age-related inflammation may also play a role in MDS development in elderly patients. MDS can be preceded by a condition known as clonal hematopoiesis of indeterminate potential (CHIP), in which specific clones carrying pre-leukemic mutations are stably over-represented in the blood in the absence of a hematological disease . The genh as MDS .Tet2-mutated clone [Tet2missense mutation frequently found in individuals with CHIP was recently generated. Using sc-RNAseq profiling, it has been shown in this animal model that MDS/acute myeloid leukemia (AML) phenotype progression in aged animals correlates with an enhanced inflammatory response and the emergence of an aberrant inflammatory monocytic cell population [Tet2mediated hypermethylated sites were identified on the PU.1 enhancer, driving the dysregulation of PU.1 expression and subsequent clonal expansion during aging [A common theme coming from recent studies is the association of CHIP with chronic inflammation or immune activation, such as associations between CHIP and various pro-inflammatory cytokines ,16. Moreed clone . On the pulation . Furtherpulation . From a pulation . Using tng aging .Dnmt3a, the gene most commonly mutated in CHIP [Dnmt3a−/− HSCs and 90% Dnmt3a+/+ HSCs. Subsequently, these mice were infected with Mycobacterium avium, which triggered an IFNγ-mediated immune response that typically depletes normal HSCs. Remarkably, within this experimental framework, there was a significant expansion of both phenotypically and functionally defined Dnmt3a−/− HSCs and multipotent progenitors (MPPs) only in the mice exposed to M. avium. Interestingly, a similar effect was also observed in the mice lacking one functional copy of Dnmt3a, resembling the situation observed in humans with Dnmt3amutations [On the same vein, interferon gamma (IFNγ)-signaling induced during chronic inflammation has been demonstrated to lead the proliferation of HSCs carrying loss-of-function mutations in  in CHIP   are essential components of the innate immune system, recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Aberrant TLR signaling has been observed in MDS patients, leading to the production of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1-beta (IL-1β), and interleukin-6 (IL-6), which contribute to the inflammatory microenvironment in the bone marrow . GeneticMore recently, a mouse model with increased TRAF6 expression specifically in hematopoietic cells (Vav-Traf6-YFP) was used to mechanistically deconvolute the role of TRAF6 in MDS . The RNAOn the other hand, when the mice with increased TRAF6 expression levels were subjected to an inflammatory stimulus like lipopolysaccharide (LPS), a component of bacterial cell walls that acts to induce chronic inflammation, there was a notable increase in TRAF6-overexpressing HSCs, leading to enhanced myeloid cell production when compared to that of the untreated mice  Table 1Table 1. NLRP3  is an essential component of the inflammasome, which is a multi-protein complex involved in the regulation of the immune response. In the context of MDS, it is well established that dysregulation of NLRP3 inflammasome activation plays a role in the pathogenesis and progression of the disease. The aberrant activation of NLRP3 has been associated with increased inflammation and cytokine production, potentially contributing to the altered hematopoiesis and immune dysfunction characteristics of MDS .Moreover, the current research on the activation of the NLRP3 inflammasome pathway in MDS has elucidated how a self-sustaining cycle of sterile inflammation can result in progressive pyroptosis and impairment of the hematopoietic niche . SpecifiMDS and secondary AML (sAML) present unique challenges related to their complex differentiation hierarchies and similarities between malignant and normal cells in the ecosystem . The celTraditional bulk RNA sequencing techniques have provided valuable insights into the transcriptional profiles of different hematopoietic populations. However, they fail to capture the heterogeneity and transcriptional dynamics at the single-cell level. In recent years, experimental methods for analyzing single cells, such as by single-cell genomic sequencing and single-cell RNA-seq (scRNA-seq), has emerged as a powerful tool used to dissect cellular heterogeneity, lineage commitment, and regulatory networks during hematopoiesis.Recent studies have indicated that precise clonal trajectories in MDS progression are made possible via single-cell genomic sequencing. In parallel, sc-RNAseq approaches have been increasingly used to address questions related to developmental hierarchies and interactions between malignant and immune cells. A milestone in this field was the use of single-cell transcriptomic and genotyping to parse heterogeneous AML ecosystems .Furthermore, advancements in computational techniques used to decode the composition of distinct cell types within the tumor microenvironment have recently emerged. These innovative methods leverage bulk gene expression profiles to identify and characterize different cell populations, offering a comprehensive view of the complex cellular landscape in hematopoietic disorders.The potential of single-cell sequencing techniques to elucidate hematopoiesis in physiological conditions and MDS at the single-cell resolution has been recently reviewed elsewhere . In brieIn the classical model supported by lineage-tracing studies on mice , human hBy optimizing nanowell-based technology for high-throughput scRNA-seq and genotyping, over 30 thousand cells from AML patients and healthy donors were analyzed. These data were integrated into a machine learning classifier, which successfully distinguishing malignant from normal cells. In this way, six malignant AML cell types aligned along the hematopoietic stem cell-and-myeloid differentiation axis were identified . This inIn the case of the clonal evolution of MDS to sAML, which is driven by the expansion of clones with leukemia-driver mutations, understanding clonal heterogeneity and mutation frequency is crucial for predicting the clinical outcomes. Recent single-cell sequencing studies of MDS and sAML bone marrow have unveiled distinct differentiation hierarchies linked to specific oncogenic drivers ,27. ImplOn the same vein, a recent study has revealed previously unrecognized cell populations within MDS bone marrow, providing insights into the underlying mechanisms of the disease . In thisThe inflammatory microenvironment in the bone marrow of MDS patients plays a crucial role in disease pathogenesis. The bone marrow stroma consists of various non-hematopoietic cells that support HSC functions during normal conditions and tissue repair. However, some stromal cells have been shown to directly contribute to myelodysplasia or leukemia development and can be altered during leukemia progression to promote tumor growth. In response to inflammation, stromal cells and the bone marrow microenvironment undergo alterations in their secretory profiles, promoting the survival and proliferation of MDS-initiating cells. Furthermore, the interactions between HSCs and the inflammatory bone marrow microenvironment are critical in shaping HSCs’ behavior. Inflammatory signals can alter the expression of adhesion molecules and chemokine receptors on HSCs, affecting their interactions with the bone marrow niche. The disruption of these interactions can lead to the egress of HSCs from the bone marrow and their mobilization into the bloodstream, impairing their ability to support proper hematopoiesis.In studies characterizing the bone marrow stroma, the sc-RNAseq approach was fundamental to identifying distinct cell populations in the stem cell niche, regulating hematopoietic regeneration and potentially initiating leukemia . In thisInterestingly, sc-RNAseq technology has recently been used to elucidate the effect of inflammation on the composition of the BM immune microenvironment and clinical outcomes in AML . This noThough scRNA-seq has been instrumental in understanding transcriptional dysregulation in MDS, uncovering the changes in gene expression profiles and signaling pathways, its high costs make it impractical for studying the large patient cohorts needed to effectively manage MDS heterogeneity. In parallel, bulk RNA sequencing (bulk RNA-seq) has been widely used to study gene expression profiles in heterogeneous samples. However, interpreting bulk RNA-seq data can be challenging due to the presence of multiple cell types, each with distinct gene expression signatures. This cellular heterogeneity, especially in hematopoietic tissue, hinders the identification of specific gene activity in individual cell types.To address this issue and achieve the more accurate molecular profiling of complex biological samples, the inference of relative cellular composition has emerged as a powerful tool. In recent decades, various computational techniques have been developed, employing deconvolution methods to disentangle cell mixtures and calculate the relative proportions of the different cellular components .These methods utilize computational algorithms to estimate the proportions of different cell types within bulk RNA-seq samples by analyzing the observed gene expression patterns. In simpler terms, deconvolution algorithms break down a mixture of various cell types into their individual components and calculate their respective proportions or ratios. In some cases, these algorithms can also calculate the overall expression signal of genes in each cell types. In addition, to validate the accuracy of deconvolution predictions, experimental validation using independent techniques like flow cytometry or single-cell RNA-seq can be performed.The fundamental principles of deconvolution methods rely on using reference expression profiles or reference datasets that represent distinct cell types . There aThus, the significant limitation of most deconvolution approaches is the requirement for a reference profile of cell-type expression. The choice of reference has been found to be more crucial than the methodology itself is in determining deconvolution performance . HoweverTo date, increasingly computational methods have been developed to infer cell type proportions from bulk transcriptomics data. For example, CIBERSORT (Cell-type Identification By estimating Relative Subsets Of known RNA Transcripts) uses the support vector regression algorithm to deconvolute the bulk gene expression profiles (GEPs) into cell type compositions (CTCs) based on a reference matrix that comprises the gene expression signatures (GES) of cell types of interest ,56. OtheIn the context of MDS, deconvolution approaches are becoming increasingly valuable. This is due to the availability of a growing number of RNA-seq datasets generated from different subsets of MDS patients, making them a valuable resource for understanding MDS heterogeneity . In addiRecently, the potential significance of identifying CTCs in the bone marrow of patients to predict the outcome of AML was explored . In thisIn the context of MDS, a recent study applied CIBERSORTx to assess the relative percentages of immune cells in the bone marrow of 316 patients with primary MDS [Inflammation has emerged as a significant player in MDS pathogenesis and progression. Inflammatory signals from the bone marrow microenvironment impact hematopoietic stem and progenitor cells, disrupting normal hematopoiesis and contributing to disease progression . A commoscRNA-seq studies on MDS bone marrow have been instrumental in discovering previously unknown cell populations and subpopulations, providing valuable insights into the underlying mechanisms of the disease. These studies have unveiled abnormal hematopoietic stem and progenitor cells, dysplastic erythroid and myeloid lineages, as well as dysregulated immune cells, shedding light on the cellular changes driving MDS progression.However, it is essential to keep in mind that scRNA-seq generates a static snapshot of the transcriptional landscape, which limits its ability to provide conclusive information on the dynamic changes during cell state transitions.Single-cell RNA sequencing studies have also been valuable in understanding the role of an inflamed tumor microenvironment in myeloid malignancies, revealing distinct inflammatory cell subsets associated with a poor prognosis. Nevertheless, the complexity of the data presents computational challenges due to inherent noise and nonlinearity. Analyzing such complex data requires advanced mathematical methods beyond conventional statistics, making the mapping of complex tissues like the bone marrow a challenging task that demands expertise in both experimental and computational techniques.Deconvolution methods applied to bulk RNA-seq data have been highly beneficial in unraveling the cellular composition and heterogeneity of hematopoietic tissue. These computational approaches allow us to extract meaningful biological insights and explore the dynamics of different cell types by identifying cell-type-specific gene expression patterns. Despite their advantages, there are still important limitations that can impact analysis, especially in the context of MDS. Among these, the similarity between different cell types can affect the accuracy of deconvolution methods, particularly when a specific cell type is present in small proportions compared to those of others. However, ongoing progress in deconvolution methods is expected to address and overcome these limitations, leading to more robust and accurate analyses. In the near future, the development of innovative deconvolution approaches has the potential to enable clinicians to extract valuable insights from the increasing number of bulk RNA-seq datasets from MDS patients. These advancements might facilitate the more precise molecular profiling of such complex and heterogenous biological samples.


Optimizing viral vectors and their properties will be important for improving the effectiveness and safety of clinical gene therapy. However, such research may generate dual-use insights relevant to the enhancement of pandemic pathogens. In particular, reliable and generalizable methods of immune evasion could increase viral fitness sufficient to cause a new pandemic. High potential for misuse is associated with (1) the development of universal genetic elements for immune modulation, (2) specific insights on capsid engineering for antibody evasion applicable to viruses with pandemic potential, and (3) the development of computational methods to inform capsid engineering. These risks may be mitigated by prioritizing non-viral delivery systems, pharmacological immune modulation methods, non-genetic vector surface modifications, and engineering methods specific to AAV and other viruses incapable of unassisted human-to-human transmission. We recommend that computational vector engineering and the publication of associated code and data be limited to AAV until a technical solution for preventing malicious access to viral engineering tools has been established. In vivo gene therapy holds great promise for treating many genetic disorders. Several gene therapy products have been licensed to date, allowing the treatment of previously incurable genetic diseases . Many ofHowever, certain biotechnologies exhibit “dual-use” potential, as they may inform and enable pathogen engineering by malicious actors . ManaginWhile directly enhancing the virulence of a known pathogen poses the most obvious potential for misuse, viral vector research may generate insights relevant to pathogen engineering that contribute to lowering the barrier for viral engineering and synthesis. In a world where many viruses are constrained by pre-existing immunity or suboptimal adaptation to human receptors, genetically encoded methods of immune modulation or targeted tissue tropism could plausibly transform an innocuous virus into the cause of a new pandemic. Indeed, efforts to engineer viral immune evasion were part of at least one historical biological weapons program . InsightHere, we highlight areas of viral vector research that may pose risks of enhancing or generating novel pandemic pathogens, including (1) universal genetic elements for immune modulation, (2) capsid engineering for antibody evasion, and (3) general-purpose viral engineering methodologies.To evade immune responses to vectors and transgene products, researchers are exploring mechanisms to genetically engineer viral vectors to modulate the induction of host immune responses through the insertion of distinct genetic elements. For instance, to prevent detection of viral nucleic acids by intracellular sensors, Chan et al. inserted short non-coding DNA sequences into the AAV genome to directly antagonize the activation of the TLR9 immune sensor and “cloak” the much larger AAV DNA sequence from detection . SimilarVectors such as AAV and adenovirus serotype 5 (Ad5) are limited by widespread pre-existing neutralizing antibodies , a drawbWhile insights specific to AAV feature little dual-use potential, more accessible and powerful methodologies inspired by its successes may be applicable to viruses with pandemic potential. General-purpose viral engineering methodologies that facilitate higher-throughput or more precise experiments, or that reduce or ameliorate the need for such experiments, could significantly lower the barrier to the generation of novel pandemic agents.For example, modular and efficient directed evolution methods such as the use of barcoding to achieve high-throughput multiplexed phenotypic analysis of AAV capsids can select for useful viral vector properties, including immune evasion . SimilarForeseeable future advances could greatly reduce or even obviate the laboratory expertise and equipment currently required for pathogen enhancement. The use of computational viral vector engineering in AAV capsid design was pioneered in 2019 when Ogden et al. used data from a multiplexed phenotypic analysis of an AAV capsid library to train a model for optimizing multiple properties including virus production, immune evasion, thermostability, and biodistribution . Since tWhile the flexibility and accessibility of computational engineering approaches is highly promising for the therapeutic development of AAVs, these papers highlight their potential applicability to other proteins and viral engineering challenges , potentiThe easiest way to improve vector effectiveness for clinical gene therapy while minimizing dual-use risks is to prioritize methods that are not genetically encodable. For instance, transient pharmacological immune modulation may represent a practical and low-risk alternative to vector engineering for immune modulation. Strategies explored to date include the administration of antibodies against CTLA-4 or pro-inflammatory cytokines, the use of small-molecule proteasome inhibitors, or peripheral induction of immune tolerance to capsids and transgenes with rapamycin –27. Non-Transitioning to non-viral delivery methods would dramatically reduce the potential for gene therapy research to inform the engineering of pandemic pathogens. The development of non-viral delivery methods does not directly generate or spread new capabilities for viral engineering, and may hence feature the least dual-use potential compared to viral vector approaches Fig.  31]. On. On31]. The application of computational protein engineering to virus entry, not just capsid or envelope glycoprotein immune evasion, could also be used to generate or enhance pathogens with pandemic potential. To safeguard the transformative potential of this technology and protect human health , developEnhanced AAV is not concerning in itself as it requires a helper virus for human-to-human transmission. However, the tools developed for the engineering of AAV feature dual-use potential if applied to other viruses. A first important step should be to limit the development and application of models for viral engineering to the least concerning agents, including AAV and other viruses incapable of unassisted human-to-human transmission, until the establishment of a mechanism capable of ensuring that code and training datasets are only accessible for legitimate research purposes. This is critical because sharing code for the modification of viral properties of viral families with pandemic potential irreversibly lowers the barrier to the enhancement of the respective pathogen. Deliberations on a strategy for non-physical viral engineering tools that balances accessibility and mitigation of misuse should be initiated by the U.S. Office of Science and Technology Policy in conjunction with the National Security Council, and equivalents in other nations, to avoid the conflicts of interest faced by health agencies. In conjunction, open-source platforms such as GitHub and Open Science Framework should proactively champion biosecurity by restricting access to general-purpose code that could plausibly be used to generate or enhance potential pandemic pathogens.Our findings underline the importance of mitigating biosecurity risks in fields such as gene therapy, vaccinology, and oncolytic virus research, which have not traditionally been associated with such concerns. While some countries, including the United States, have nascent policies to regulate dual-use risks, such frameworks currently don’t capture risks from insights transferable between agents, nor from technologies outside of biology, and are typically implemented by agencies lacking security expertise that directly sponsor the relevant research , 38. To Gene therapy holds enormous promise for human health. However, some lines of viral vector research may make it substantially easier to generate or enhance pandemic pathogens. To minimize this risk, we recommend preferentially advancing lines of research with less potential for misuse. When methods with dual-use potential are pursued, effective solutions to mitigate misuse must be created. In a time of increasing biotechnological capabilities accessible to a growing number of individuals, proactively mitigating the potential for well-meaning research to be misused will protect human health and safeguard future biomedical advances.


Using proton nuclear magnetic resonance, we identified 136 metabolites among participants. Our non-hypothesis-based approach identified distinguishing urine metabolic features in the high-risk group, revealing increased levels of hippurate and other gut-derived metabolites and decreased metabolites related to central energy metabolism when compared to referent groups. Our complementary hypothesis-based approach, focused on nicotinamide adenine dinucleotide (NAD+) related metabolites, and revealed a higher kynurenate/tryptophan ratio in the high-risk group (p = 0.001), consistent with a heightened inflammatory state. Workers in high-risk occupations are distinguishable by urinary metabolic features that suggest increased gut permeability, inflammation, and altered energy metabolism. Further study is needed to explore the pathophysiologic implications of these findings.Mesoamerican nephropathy (MeN) is a form of chronic kidney disease found predominantly in young men in Mesoamerica. Strenuous agricultural labor is a consistent risk factor for MeN, but the pathophysiologic mechanism leading to disease is poorly understood. We compared the urine metabolome among men in Nicaragua engaged in sugarcane harvest and seed cutting ( Mesoamerican nephropathy (MeN) is a devastating form of chronic kidney disease (CKD) in agricultural communities in the Pacific coastal region of Mesoamerica . Two decEnvironmental stressors linked to agricultural labor are of particular interest in MeN, given the high prevalence of the disease among agricultural workers. Through use of metabolomics, the study of patterns of small molecules derived from cellular metabolism and exogenous sources , we can An area of particular interest regarding the pathophysiology of MeN is derangement in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). NAD+ is an essential coenzyme linking glycolysis and the citric acid  cycle with the mitochondrial electron transport chain ,8. NAD+ Alterations in energy metabolism centered around NAD+ emerge in the setting of physiologic stress in the kidney. These alterations are characterized by a decline in cellular NAD+ biosynthesis mediated in part by decreased activity of quinolinate phosphoribosyltransferase (QPRT) ,10. DeplNAD+ biosynthesis occurs along three key pathways: the de novo pathway that utilizes tryptophan (Trp), the salvage pathway that uses nicotinamide (Nam), and the Preiss–Handler pathway that uses the acid form of Nam, nicotinic acid  7]. In . In 7]. 1H NMR) to characterize the urinary metabolic features of men in Nicaragua working as sugarcane harvest or seed cutters, job categories at very high risk for MeN based on prior research  + 0.705, 95% CI of slope = 0.119 to 0.291, Results of heatmaps and mean decrease accuracy assessment from random forest comparisons across groups are shown in 2, findings were largely similar to the original analysis for the CS vs. OW and CS vs. NW comparisons (When we repeated non-hypothesis based PLS-DA and pathway analyses in subset of individuals with an eGFR ≥ 90 mL/min/1.73 mparisons . In the In this cross-sectional analysis of workers undertaking manual labor in Nicaragua and Spain, we observed that distinct urinary metabolic features distinguished our pre-specified high-risk group, Nicaraguan sugarcane harvest and seed cutters, from other occupational groups with respect to both the urinary metabolome as a whole and NAD+ metabolism in particular. One of the most striking differences between the high-risk CS group and other occupational groups was the degree of urinary hippurate elevation observed. Elevated hippurate may reflect increased gut permeability in the high-risk group. Prior study of the urine metabolome of heat-stressed goats demonstrated that elevated hippurate levels are a fundamental determinant of between-group differences, in conjunction with increases in other gut-related metabolites . Our stuComplementing these findings, in hypothesis-based analyses urinary KynA/Trp was increased in the CS group compared to referent groups, again consistent with a greater inflammatory state associated with high-risk work ,34. PrioIncreased urinary hippurate could reflect other processes as well. Increased urinary hippurate is associated with increased renal blood flow . UrinaryIn our investigation of the NAD+ biosynthetic derangement, urinary NAD+ trended higher, rather than lower, in the high-risk CS occupational group, and urinary NAD+/Trp was increased rather than decreased. Unfortunately, the urinary Q/T ratio, a better validated marker of NAD+ biosynthetic derangement ,11, was Patterns in TCA cycle intermediates, which tended to be lower in the CS group, further support the conclusion that NAD+ elevations in this study may not be a product of increased central energy metabolism. We observed decreased metabolites related to alanine/aspartate/glutamate metabolism, which is involved in the catabolism of proteins for energy generation by cellular mitochondria as an alternative input to the TCA cycle . SimilarTCA cycle components, such as citrate, 2-oxoglutarate, and fumarate are dicarboxylates, so decreased levels could alternatively reflect differences in sodium-dependent proximal tubular reabsorption, with increased sodium reabsorption associated with increased dicarboxylate reabsorption largely mediated through the sodium-dicarboxylate cotransporter NaDC1 . Given MAnalysis of the urine metabolome enabled us to distinguish the high-risk CS group from other groups with reasonable accuracy using random forest. This accuracy was generally lower when comparison groups were pooled to reflect shared risk based on region of residence, labor intensity, and work in agriculture. This suggests that metabolic elements defining the CS group are more distinct than metabolic elements defining any one shared risk factor. Increased hippurate as well as decreased TCA cycle intermediaries remained important features distinguishing groups, while features related to consumption, such as caffeine and proline betaine were key features distinguishing Spain and Nicaragua-based groups . OverallDifferences existed between the CS group and referent groups beyond the designed differences in work intensity, residence, and an agricultural work environment. These should be considered when assessing the findings presented here. The SW and NW groups were older on average than the CS group. Unlike for the CS group, specimens for the NW group were not universally collected at the end of a work day. The SW group also had worse kidney function on average than the CS group, and included more individuals with proteinuria and self-reported hypertension and fewer individuals with weekly NSAID use.Another important consideration in interpreting the results of this study is that differing urinary metabolite levels may result from alterations in metabolism, alterations in transport from intracellular to extracellular spaces, and alterations in the kidney’s handling of those metabolites, all of which we are unable to differentiate between due to our observational, cross-sectional design. We did not evaluate how strenuous each individual’s labor was on the day of specimen collection, and it likely differed among individuals within the same occupational groups, potentially blurring the distinction in intensity of manual labor among groups. All groups engaged in some amount of manual labor, further limiting differences in this exposure between comparators; the NW group was composed of brickmakers and miners, both occupational groups that are also considered to be at increased risk for MeN ,27. PartOur findings suggest several areas for further exploration. These include future reevaluation of these study participants to determine who went on to develop chronic kidney disease, which can serve as a crucial additional comparison to understand whether certain metabolic features may either predispose to subsequent CKD development, or be biomarkers of early disease prior to decline in glomerular filtration rates. In addition, evaluating pre-, during-, and post-work specimens from high-risk groups would enable a more comprehensive determination of metabolic changes arising over the course of a work shift.Features in the urinary metabolome of Nicaraguan sugarcane harvest and seed cutters differentiated them from other groups of workers performing manual labor. A key distinguishing factor was their elevated levels of hippurate and other gut-derived uremic metabolites. In addition, sugarcane harvest and seed cutters showed evidence of increased KynA generation, which may indicate the presence of a greater inflammatory state in these individuals. Taken together, our findings support the hypothesis that inflammation from increased gut permeability due to heat stress may be present in this high-risk occupational group. Other pathways distinguishing cane cutters and seeders from referent groups suggest there may be further physiologic differences, including alterations in NAD+ metabolism, central energy metabolism, and proximal tubular transport function. This study is among the first to date to explore the urinary metabolome among individuals at risk for MeN, and demonstrates the potential of metabolomics as a tool to explore the pathophysiology of this still incompletely understood disease.