Attenuated and/or genetically modified oncolytic viruses (OV) gain increasing interest as a promising approach for cancer therapy. Beside the assessment of subject safety, quality and efficacy aspects of medicinal products for human use, genetically modified viruses are also governed by EU regulatory frameworks requiring an environmental risk assessment (ERA). An important element to be assessed as part of the ERA is the incidence of exposure to OV of individuals, other than the trial subjects, and the environment. The evidence-based evaluation of shedding data is considered to be decisive in that context, as it may impact the OV capacity to be transmitted. This is particularly true for OV still able to  replicate as opposed to replication-defective viral vectors commonly used in gene therapy or vaccination. To our knowledge, this article presents the most extensive and up-to-date review of shedding data reported with OV employed in clinics. Besides the identification of a topical need for improving the collection of shedding data, this article aims at providing an aid to the design of an appropriate shedding study, thereby relying on and further complementing principles described in existing guidelines issued by European and international institutions. Oncolytic viruses (OV) are  replication competent viruses (with low pathogenicity) that are designed to be able to selectively replicate in tumor cells, leading to their destruction by the direct lysis of host tumor cells, while sparing normal cells. Accumulating evidence in oncovirotherapy demonstrates that OV infection can also trigger specific antitumor immune effects. Cellular proteins released from OV-lysed tumor cells elicit an interaction with the innate immune system through the activation of dendritic cells, which in turn stimulate adaptive immunity .OV can be naturally occurring viruses that have natural tropism to neoplastic cells, such as Reoviruses , Newcast®® , an oncolytic Herpes Simplex Virus (HSV), for the treatment of advanced melanoma can be considered as a milestone in the development of OV therapy, it should be noticed that three other OV have been approved for commercialization. A wild-type Enteric Cytopathogenic Human Orphan type 7 (ECHO-7) Picornavirus, with trade name Rigvir®®, was first approved in 2004 in Latvia for the treatment of melanoma [®®) received conditional approval in Japan for the treatment of glioblastoma [While the approval in the US and Europe in 2015 ,8,9  as part of the regulatory application for clinical trials or marketing applications of medicinal products containing genetically modified organisms (GMO).The evaluation of exposure pathways through which GM viruses and viral vectors with their inserted DNA sequence may interact with humans , or the environment, is of major importance in the ERA. Pathways of exposure include leakage upon administration (spreading) of the investigational product, accidental release during its administration, and shedding of the product by the trial subject. Shedding corresponds to the dissemination of viral (vector) particles in any form into the environment via excreta ), skin  and blood from the treated subject . Unlike While several guidelines are recommending to examine shedding as early as possible in the clinical development (see hereunder), in practice, rather limited attention is given to shedding data compared to the relatively extensive collection and assessment of data supporting the safety, quality and efficacy assessment of the OV.This paper presents a review of shedding data collected during the clinical development of OV and aims at providing an aid in the design of an appropriate shedding study.Based on general reviews on OV recently published in PubMed ,20, a fiThis final list counts 165 manuscripts representing 165 different clinical trials and each trial was evaluated for multiple variables. Variables assessed included the phase of the clinical trial, the number of subjects treated, the type of virus used, the nature of the viral backbone , the transgene, the type of cancer treated, the use of single-agent or combination regimens and viral genome shedding data. With respect to the viral genome shedding, the authors assessed for each of the studies whether viral genome shedding was assessed and reported, and more particularly, which body tissues and/or fluids were assessed for viral particles, which detection test was used, whether viral particles were detected and for how long, and whether shed particles contained infectious viral particles. This final list of 165 manuscripts can be found in online In addition to the regulatory requirements common to all  medicinal products, the use of GM viruses for pre-clinical investigation, clinical trials as well as their marketing as medicinal product is covered by the legislation regulating the use of GMO, which encompasses an ERA.The ERA relies on a well-defined methodology and should be conducted on a case-by-case basis. It starts with the identification and characterization of potential hazards associated with the GMO on human health, with focus on individuals other than subjects or those vaccinated, and on the environment at large, including animals, plants and microorganisms. Concurrently and as part of a second risk component, the probability of occurrence of potential hazards under the conditions of use is assessed. Both components led to an estimation of the risk to human health and the environment posed by each identified hazard of the GMO by combining the probability of its occurrence and the magnitude of its consequences. An overall risk is then determined by combining all of the individual risks ,22. The Numerous viruses have been used to design and develop OV therapy. The identification of potential hazards, the first step in the ERA, should take into account the characteristics of the OV and the properties of the inserted gene sequences and the gene products. This implies that due consideration of aspects such as the extent of attenuation, the replication capacity, tropism, biodistribution, genetic stability and the capacity to recombine with other wild-type viruses should be evaluated on a case-by-case basis. The properties of the parental viruses from which OV are derived may provide valuable starting information, taking into account that, in general, OV developed for therapeutic application are less virulent or infectious.The development of different oncolytic viral systems for cancer therapeutic applications relies to a major extent on the genetic modification of viruses. Several strategies to enhance the therapeutic effect of oncolytic virotherapy involve the genetic “arming” of replicating viruses with transgenes, such as tumor suppressor genes, immune regulatory genes, apoptosis inducing genes, angiogenesis inhibitory genes and genes coding for pro-drug converting enzymes or heat shock proteins. Besides the insertion of transgenes with an inherent therapeutic effect, other sequences can be inserted or deleted that are involved in the targeting of OV .Inserted gene sequences and their gene products should be considered for their potential impact on the viral life cycle alterations , on the capacity of recombination of the virus or on the host . All of these elements may alter biodistribution and persistence in the subjects and may impact shedding following administration of the OV.In the next section, we will focus on elements that may have an impact on the shedding properties of OV, based on examples in the clinical field, and elaborate on the assessment of shedding data as one of the key aspects in the outcome of the ERA of OV.As mentioned above, shedding corresponds to the dissemination of viral (vector) particles in any form into the environment via the excreta or secreta, skin and blood from the treated subject . While iFrom our review of the literature see , it is oThe test method used to assess the shedding potential of oncolytic viruses should be sufficiently sensitive ,26,27. POur review of the literature shows that many studies limit the collection of shedding data by conducting quantitative real-time PCR (qRT-PCR) analyses. Of the 70 clinical trials mentioning shedding analysis, 4 did not mention the method. In 32 of the clinical trials, only a PCR test was performed, while 14 of the studies did only perform an infectivity test such as a cell culture or a plaque assay test. In 17 of the trials, both a PCR and a cell culture or plaque assay were performed.®®, for which swabs from the surface of injected lesions from subjects with unresectable recurrent melanoma tested positive for infectivity [In most of the clinical trials that performed infectivity tests alone or in combination with a PCR, no infectious particles were observed in the various shedding samples analyzed. However, in a few trials 6/32), the presence of infectious particles was shown by cell culture or plaque assay in some shedding samples. This was the case for some saliva samples from metastatic prostate cancer subjects treated with a high dose of the oncolytic Adenovirus CG7870 , and forectivity , as wellectivity . We also, the preectivity . All theA proper ERA of OV addresses several aspects that may impact the release of the viral particles by the host. This includes among others an examination of the genetic stability, the conditions under which replication is occurring, replication competence and the route of administration of the OV. Characteristics from the wild-type virus from which the OV has been derived, such as the pathogenicity, the tropism, the host range, the natural route of transmission and the shedding pattern, may provide valuable information to perform the ERA of OV. Furthermore, each of these aspects may be altered by the overall design and the genetic modification proper to a given OV ,34.®® [®® and OrienX010 both express the transgene GM-CSF used to boost the anti-tumor immune response by promoting dendritic cell recruitment and activation following tumor antigens’ liberation from lysing tumor cells. Although OH2 also expresses the transgene GM-CSF, no shedding of viral particles has been observed [With regard to the type of virus from which OV is derived, the detection of a viral DNA/RNA genome was observed at least once for all virus families . A rathe®®  and Orie®® . No shed®® , G207 [3®® ,38,39, G®® , HF10 [4®®  or HSV17®® ,43. Imlyobserved .Differences in shedding pattern have also been observed within the Picornaviruses class. Shedding of the viral particles from subjects treated with the naturally occurring replication-competent Picornavirus, Coxsackievirus  or SenecIn addition to the intrinsic properties of the OV and irrespective of the type of virus, due account should be given to the interaction of the OV with the host, which may have an effect on this interaction and hence on the shedding pattern. One should therefore remain cautious with extrapolating pre-clinical data to human beings. For example, the results of quantitative real-time PCR (qRT-PCR) indicate that VSV-IFNβ-NIS RNA was detectable in some early nasal, oral, and rectal swabs of inoculated pigs , or in sThe route of administration used during clinical application usually differs from the natural portal of entry of the wild-type virus from which the OV are derived, and may also impact shedding pattern A,B. IntrThe same observation can be made with two oncolytic Poxvirus GL-ONC1 and Pexa-Vec. Shedding was observed when GL-ONC1 was delivered intravenously to subjects with advanced head and neck carcinoma , whereasFor oncovirotherapy, direct intratumoral injection is a preferred route of administration for brain tumors (18/23), despite the fact that it may pose significant challenges. Intratumoral injection of the OV is also mainly used for easily injectable tumors, such as melanoma (16/22), or head and neck cancers (6/8) C.OV are also often administered intravenously in subjects with metastatic tumors. In this case, OV encounter many physiological barriers before reaching cancer lesions and repeated doses may be necessary, thereby triggering the recognition and attack by the immune system and clearance of the OV by neutralizing antibodies. While the latter is a concern for efficacy reasons, elicitation of a strong immune response may also result in a shorter duration of shedding.As has been observed by Dunn et al.  and WeilFinally, the reactivation of latent OV, influenced by the immune status of the subject, could possibly lead to a shedding pattern that is different from what could have been predicted from former clinical experiences. For oncolytic viruses with the potential for latency reactivation, the collection of additional samples for shedding analysis when subjects show signs of infection due to reactivation could be planned.Each of the above-mentioned elements contributes to a risk-based approach in the design and the extent of shedding studies. It may provide insights into the choice of biological samples to be analyzed, as well as the frequency and the duration of monitoring.Samples most commonly collected include urine and oral swabs. Other sample types such as feces, nasal swabs and injection site samples are also collected, but less commonly see .The collection of data should also take into account the occurrence of metastasis. For example, secondary tumors may also be located in the oral cavity, larynx, pharynx or esophagus, justifying the collection of saliva samples.®® [®® DNA was observed on the surface of injected lesions for all treated subjects, and it was still detected for 14% of subjects during the safety follow-up period. Of the 740 swabs from the surface of injected lesions, 8 tested positive for infectivity [When OV are administrated by the intradermal route, there is a possibility of transmission of the OV through skin contact. Therefore, the collection of skin swabs at the site of injection, in addition to samples routinely assessed for shedding, should be recommended in order to determine whether an appropriate occlusive dressing is required as a precautionary measure. According to our analysis, none of the trials performing intradermal injection reported shedding results. On the other hand, eight trials where the OV were administrated intratumorally included swabs from the injection site in their shedding analysis. From these eight trials, shed viral DNA at the injection site was observed in five trials. Of these five clinical trials, four trials were performed with Imlygic®® ,64,65,66®® . Detectaectivity . The Adeectivity .The duration and frequency of sample collection should be decided on a case-by-case basis, taking into account the characteristics of the parental virus, the conditionally replicative competence of the OV and the immune status of the subject. As most of the OV are still replication-competent, the duration of sample collection should also take into account the possible appearance of a secondary peak of shedding. As one of the numerous examples of viral replication, it was shown that in subjects treated with the intratumorally injected HSP70, a telomerase-specific replication-competent oncolytic Adenovirus (telomelysin), viral DNA was detectable in plasma or sputum at days 7 and 14 post-treatment, despite being below detectable levels at 24 h post-treatment .The identification and characterization of potential adverse effects associated with the use of a given OV and the assessment of their likelihood of occurrence may lead to the identification of potential risks. It is also possible that the case-specific assessment reveals remaining uncertainties, precluding any conclusion on the risks for human health and the environment. In the first case, the set-up and implementation of risk management measures aim at minimizing identified risks, while in the second case, these could serve as precautionary measures.In cases wherein data support the low likelihood of shedding or where no risk related to the shedding is identified for human health or the environment, good working practices involving proper hand hygiene, personal protective equipment (PPE) and proper decontamination and waste procedures at the clinical setting will be effective to limit the inadvertent exposure of personnel and the possible dissemination of the OV into the environment . The corWhen shedding by the treated subject cannot be excluded and potential risks for human health and the environment have been identified, risk management measures should also focus on minimizing the exposure of third parties outside the clinical setting, thereby giving particular attention to immunosuppressed or any vulnerable people . A list of possible measures that may be considered, taking into account the considerations made as part of a case-specific risk assessment, is provided in a guidance document endorsed by several national competent authorities involved in the risk assessment GMOs  and has Geletneky et al. indicated in their article that glioblastoma subjects treated with the oncolytic Parvovirus H-1 were obliged to stay isolated in the study center until shed viral genomes were no longer detected in feces, urine or saliva, or until the first occurrence of H-1-specific antibodies .It could be hypothesized that OV applications that have entered the market and that have successfully gone through the several stages of clinical development would rely on shedding data to determine risk management measures, if deemed relevant according to a proper risk assessment, and that data on the occurrence of transmission to close contacts would allow further refinement of risk management measures. In this ideal scenario, evidence-based data would contribute to a set of management measures that are as proportionate as possible to the environmental risk. However, the occurrence of secondary infections is barely addressed during clinical development. Given that these viral vectors are often derived from infectious viruses, any studies that contribute to the assessment of possible secondary infection would further contribute to the ERA of the medicinal product.®®, Andtbacka et al. reported that some mucosal or skin lesions were observed in close contact, and investigators all tested negative for T-VEC. However, none of the lesions were tested for wild-type HSV-1 or HSV-2, leaving an uncertainty as to the origin of the lesion [During a phase II clinical trial with modified Herpesvirus Imlygice lesion .®® are also found in the current subject brochure for Imlygic®®, which stipulates measures to be taken by the subject to avoid the direct contact of thirds with the subjects’ bodily fluids or injection sites, such as covering injection sites with airtight and watertight dressings, implementing a proper disposal of used dressings and cleaning material to prevent household contacts from directly touching them [The precautionary measures that were implemented for the subject during this phase II study with Imlygicing them .®®, started in 2017, is among others counting the numbers of herpetic infections, with the detection of T-DNA among close contacts and healthcare providers as a secondary outcome [®® are being pursued.An ongoing post-marketing study of melanoma subjects treated with Imlygic outcome . This exFinally, if animals may be infected, appropriate measures to limit exposure to susceptible pets or other animals in the immediate surroundings of the treated subject should be considered. Newcastle disease virus (NDV), for example, is a naturally occurring oncolytic virus that causes severe illness in birds and poultry, and could therefore pose an environmental risk even if it is non-pathogenic in humans. In three clinical trials with intravenously injected NDV-PV701 in subjects with advanced solid cancer ,72,73, lAnother example of clinical trials where measures to avoid contact with animals have been proposed involved the use of a recombinant chimeric Vesicular stomatitis virus, in which the VSV glycoprotein was replaced to abrogate neurotoxicity and pathogenicity . As the The inventory presented in this paper provides a state-of-the-art of the shedding analysis of OV in clinical practice, and raises the question: to what extent it is possible to build upon the experience gained so far with shedding data to draw conclusions for each of the different types of OV? This may be particularly relevant in cases where historical shedding data have been obtained for well-characterized OV, of which the designs present relevant similarities with a novel investigational OV. Our literature search reveals that in 89% of the studies with Adenovirus-derived OV reporting shedding analysis, positive shedding results were obtained. However, as illustrated above by the several examples developed in this paper, the shedding patterns remain diverse and complex, as well as for Adenovirus-derived OV, thereby hindering the development of a standardized study design. Diversity in shedding patterns was observed even when using OV derived from the same type of virus. A number of elements may impact the shedding pattern, such as the specific design of the viral vector and the transgene harbored by the viral vector, the interaction of the OV with their host organisms, the immune status of the patient or the variety of the clinical protocols, such as differences in the administration route or the use of concomitant drugs.Valuable information and insights into the toxicity, biodistribution and shedding pattern of OV could be obtained from non-clinical studies. As compared to clinical studies, animal studies are more amenable to be conducted in the early stages of the development of investigational OV. However, it should be taken into account that data are not readily extrapolable from animals to human, in particular when different routes of administration are used or no data have been collected in larger animals, such as non-human primates. Also, an animal model fails to mimic the patient-specific immune status. This means that an absence of viral shedding in animal studies does not necessarily preclude viral shedding in humans, as a different host organism may induce a different behavior of the virus .Because the shedding pattern strongly depends on different factors and because it can also differ between animals and humans, the collection of shedding data in the earliest phase of clinical development of investigational OV is strongly recommended regardless of the OV vector.The FDA guidance for the design and analysis of shedding studies also recommends the collection of shedding data on OV in the earliest phase of clinical development (phase I) and anytime afterwards if the dose, the route of administration, the regimen or the indication are modified . LikewisA possible concern, for which we could not find experimental data, is the likelihood of an in vivo recombination between the OV and endogenous viruses circulating in the trial participants. This assessment should not be neglected because recombination events could lead to the formation of uncharacterized variants that could be more virulent and that could affect the shedding pattern and the potential for transmission. These newly generated viruses could therefore compromise the environmental safety. In general, the likelihood of the recombination between viruses significantly increases with the prevalence of co-circulating viruses in the population, and with their genetic homology. High viral loads, often a relevant feature of replication-competent OV, increase the chance of exchanging strands, explaining why, in many cases, recombination is often replication-dependent . MoreoveWhilst the collection of shedding data undoubtedly supports an evidence-based ERA, it is important to be aware that the shedding of OV or any other viral vector or virus does not necessarily involve a risk. Potential adverse effects for close contacts or the environment that could arise following shedding do not only result from the presence of viral particles in the shedding samples, but also depend on the stability of the shed viral particles under environmental conditions outside the host, the route of transmission , the capacity of the shed particles to infect cells of other persons or animals, and, as a last element in the chain of events for environmental risk to occur, the capacity of the OV to cause adverse effects in the novel host organism. To possibly alleviate remaining uncertainties in secondary transmission, it is therefore important to answer the questions of whether the observed shed particles are only vector DNA/RNA or a remnant thereof, and whether these shed particles are still infectious. These observations will contribute to a proportionate risk management by allowing the determination of appropriate precautionary measures.Indeed, a fundamental question when uncertainties remain regarding the actual risk associated to the shedding of OV is what level of risk management measures should be taken, or what level of uncertainty would warrant precautionary measures. If it remains unclear whether shedding may occur and what risks it may entail for human health and the environment, a drastic and conservative scenario would be to eliminate any possibility of release of OV into the environment by keeping patients for several weeks/months in the hospital setting. However, the implementation of stringent measures may carry drawbacks, as it may increase costs and time not only for the appropriate training of personnel, the set-up and follow-up of waste disposal procedures and logistics, but also for the recruitment of subjects requiring their informed consent and the training of close contacts if the trial participant is discharged. Ideally, risk management measures should be as proportionate as possible to the actual risk posed so as not to hinder the development of research and innovation and to safeguard patient access to innovative treatments, while ensuring the proper protection of human health at large and the environment. A way to contribute to proportionate risk management is to continue to gathering data-based evidence by including within the shedding analysis the determination of the fraction of infectious particles in early phases of the clinical development of OV.With the diversity of OV that entered the clinical research and development landscape, this work demonstrates the current gaps in data-based evidence on shedding and the challenge of defining risk management measures that are proportionate to the actual risk posed for human health and the environment. In accordance with GMO legislation requiring a case-specific and risk-proportionate approach, this paper aims at encouraging the collection of shedding data as early as possible in the developmental plan in the rapidly growing area of OV. The demonstration of the shedding of infectious particles may warrant assessments of the potential of secondary transmission. The collection of real-world transmission data is expected to provide a better understanding of transmissibility, which is key to characterizing the risk for the human population and the environment. It will also benefit future clinical trials developers in establishing a clinical protocol based on evidence-based risk assessment.


Urinalysis was remarkable for +3 proteinuria and hematuria with 11–25 red blood cells per high-power field. Urine protein/creatinine was 6.2 g/g. Patient underwent kidney biopsy, with light microscopy showing diffuse thickening of the glomerular capillary walls and mesangial expansion  3 months prior. His vitals were normal on arrival, and cardiopulmonary, abdominal, and neurologic examinations were all unremarkable. He had +2 lower extremity edema and a discoid rash over his chest and upper extremities  cause TNF-α antagonist-induced lupus-like syndrome (TAILS).1 Etanercept is a soluble, human fusion protein, which functions as a TNF inhibitor by competitively binding to TNF and preventing its activation. Unlike infliximab, there are fewer reports of etanercept inducing TAILS.2 Etanercept-induced lupus (EIL) mostly affects women, and the onset of symptoms takes a mean of 4.4 months from the start of medication.1 Symptoms of EIL usually include SLE cutaneous features such as malar rash, discoid rash, and photosensitivity. Other systemic symptoms include fever, weight loss, arthritis, serositis, and myositis. Although neurological and kidney involvement is rare, class III and IV lupus nephritis and less commonly class V are more often seen in EIL than in other DIL.4 Induction of autoantibodies (ANA and anti–ds-DNA) has been observed in patients receiving anti-TNFα agents.3 The presumed mechanism is induction of apoptosis promoting antibody production against uncleared antigenic nucleosomes.5 The diagnosis of TAILS is based on history and clinical features. Kidney biopsy will be helpful in the setting of kidney involvement. In almost all cases, the disease abates after cessation of the causative drug. However, immunosuppressive agents are required in complicated cases including organ involvement such as lupus nephritis.3Drug-induced lupus (DIL) is the occurrence of a lupus-like syndrome after exposure to certain medications. TNF-α antagonists can rarely (<1% of cases) cause TAILS. Induction of autoantibodies (ANA and anti–ds-DNA) has been observed in patients receiving anti-TNFα agents.TNF-Etanercept is a soluble, human fusion protein, which functions as a TNF inhibitor by competitively binding to TNF and preventing its activation. Unlike infliximab, there have been fewer reports of etanercept inducing TAILS.Lupus nephritis is more often seen in EIL than in other DIL.


Tumor necrosis factor-alpha (TNF-α) inhibitors have been shown to be well tolerated among patients with rheumatoid arthritis, inflammatory bowel disease, and psoriasis. Meanwhile, more recently, clinical practice and research efforts have uncovered increasing cases of psoriatic lesion development tied to initiating treatment with a TNF-α inhibitor. The underlying mechanisms associated with this occurrence have yet to be fully elucidated. A review and analysis of cases of paradoxical psoriasis currently published in the literature is warranted. In addition, exploring possible mechanisms of action and potential treatment options associated with favorable outcomes is much needed.A systematic literature review was performed utilizing PubMed and Google Scholar databases (1992-present), in which 106 cases of paradoxical psoriasis were reviewed. The most common morphology developed was plaque psoriasis vulgaris. There was a female predominance (61.3%), and the most common underlying autoimmune disease was rheumatoid arthritis (45.3%). In addition, the most commonly associated drug with the onset of psoriatic lesions was infliximab (62.3%). Furthermore, the findings suggest that the most well-supported mechanism of action involves the uncontrolled release of interferon-alpha (IFN-α) from plasmacytoid dendritic cells (pDCs) after TNF-α inhibition. While TNF-α inhibitors have been shown to have great benefits to patients with rheumatologic diseases, cases of paradoxical psoriasis demonstrate the importance of close monitoring of patients on TNF-α inhibitors to allow for early recognition, treatment, and potentially change to a different mechanism of action of the medication used to prevent further progression of the inflammatory lesions. Tumor necrosis factor-alpha (TNF-α) is a cytokine generated by activated macrophages, T-lymphocytes, neutrophils, and natural killer (NK) cells to regulate inflammatory responses. Tumor necrosis factor-alpha activates intracellular signaling pathways by binding to either TNF-receptor 1 (TNFR1) or TNF-receptor 2 (TNFR2) . Excess Due to the crucial role of TNF-α in the pathogenesis of these diseases, TNF-α inhibitors have revolutionized the treatment of autoimmune diseases, demonstrating increased efficacy compared to alternative treatments. Currently, five TNF-α inhibitors are being used. These drugs include infliximab, adalimumab, certolizumab, pegol, golimumab, and etanercept ,5. AlthoPsoriasis is an autoimmune T-cell-mediated disease of the skin characterized by sustained inflammation in the stratum corneum, which stimulates abnormal keratinocyte proliferation and differentiation . A mouseAs the inflammatory response cascades, increased circulating TNF-α results in the infiltration of inflammatory cells from the blood into the skin and dendritic cell activation. TNF-α also increases the activation of keratinocytes, promotes epidermal hyperplasia, and stimulates the nuclear factor kappa B (NF-kB) inflammatory pathway to further increase the production of pro-inflammatory cytokines . A 2004 However, increasing numbers of cases of paradoxical development of psoriasis in patients with autoimmune diseases treated with TNF-α inhibitors have been reported in the literature. The first case of paradoxical psoriasis induced by TNF-α inhibitors was reported in 2004, but with increasing usage of TNF-α inhibitors, more cases are being reported. The current incidence of paradoxical psoriasis lesions is 2%-5% . CurrentAlthough the number of cases of paradoxical psoriasis continues to increase, there is still no clear mechanism of action established and little research evaluating the treatments that result in the best outcomes for patients who develop these lesions. In this review, we aim to review the current literature regarding proposed mechanisms of action and published cases of TNF-α inhibitor-induced psoriasis. Based on our analysis, we aim to propose treatment options that will help guide clinicians in managing patients to allow for better patient outcomes with complete remission of psoriatic lesions.Proposed mechanisms of actionAlthough the mechanism of action tied to the development of psoriatic lesions has yet to be fully elucidated, there are several well-supported theories on the mechanism by which TNF-α inhibitors result in psoriatic lesion development. The most accepted theory is that the TNF-α inhibitor leads to uncontrolled production of IFN-α by pDCs. Plasmacytoid dendritic cells have been found to be increased in the pre-psoriatic skin of psoriasis patients and appear to initiate autoimmune inflammation leading to psoriatic lesion formation. Plasmacytoid dendritic cells infiltrate the skin and produce a surge of IFN-α. As a result, excess levels of IFN-α activate myeloid dendritic cells, which then stimulate pathogenic T cells. This results in the release of the inflammatory cytokines IL-23, TNF-α, and IL-12, which activate T helper cells, stimulate further inflammatory cytokine release and result in unregulated keratinocytes . A studyIn addition to the effect of TNF-α inhibitors on IFN-α and pDCs, it appears that TNF-α inhibitors may also upregulate the production of TH1 and TH17 cells. A 2014 study was the first to show that an increased number of IFN-γ-secreting TH1 and IL-17/IL-22-secreting TH17 cells were found in patients who developed TNF-α inhibitor-induced psoriasis, suggesting a potential link between TNF-α inhibitor treatment and increases in cytokines strongly associated with psoriasis, such as IL-17 and IL-22 [Furthermore, TNF-α inhibitors may also lead to abnormal lymphocyte migration and upregulated expression of CXCR3 ligands, which have been shown to be involved in the development of psoriatic lesions . MeanwhiAs opposed to classical psoriasis, it does not appear that auto-reactive T cells are involved in paradoxical psoriasis. In a T-cell-depleted mouse model, treatment with TNF-α inhibitors resulted in paradoxical psoriatic lesion development as a result of an overactive Type 1 IFN-driven inflammatory response. The paradoxical psoriatic lesions had increased dermal accumulation of pDCs, reduced T cell numbers, and higher levels of Type-1 interferon expression when compared with classical psoriatic lesions.  This datMethodsA systematic literature review was performed utilizing the PubMed and Google Scholar databases 1992-present). Search terms included "tumor necrosis factor-alpha inhibitor," "TNF-α," and "paradoxical psoriasis" combined with the terms "psoriasis," "pathogenesis," and "treatment." After considering inclusion and exclusion criteria, 18 peer-reviewed publications were identified and utilized . However, the incidence of palmoplantar pustulosis in the general population with psoriasis is less than 20% . This coWhile the exact mechanism of action that induces the development of paradoxical psoriasis has yet to be fully elucidated, several well-supported theories continue to be evaluated. The most widely accepted theory implies a key role for pDCs and their production of IFN-α in the induction of psoriatic lesions. Since TNF-α functions to inhibit pDC maturation and IFN-α, TNF-α inhibition may allow for uncontrolled and unregulated production of IFN-α, consequently initiating inflammatory pathways involved in the onset of psoriatic lesions . This idIn the cases reviewed, infliximab 62.3%) was the most reported TNF-α inhibitor to induce psoriatic lesions, with adalimumab (19.8%) being the second most common. This may be attributed to the more common usage of infliximab since it was the first TNF-α inhibitor to receive U.S. Food and Drug Administration (FDA) approval in 1998 for inflammatory bowel disease (IBD) . Whereas.3% was tBased on our analysis, we suggest that patients discontinue treatment with their TNF-α inhibitor if another reasonable treatment option for their initial disease is available. Additionally, treatment with topical corticosteroids and phototherapy may be beneficial to those patients who develop mild psoriasis in order to aid in the quicker resolution of lesions. In patients who develop moderate-to-severe psoriasis with significant impacts on quality of life, treatment with methotrexate or systemic therapy allows for an increased probability of resolution. It is important for clinicians to implement close monitoring of patients upon starting treatment with TNF-α inhibitors to allow for early recognition and rapid initiation of treatment for complete resolution of lesions.Tumor necrosis factor-alpha (TNF-α) inhibitors have proven to be effective drugs for various rheumatologic diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel diseases. While TNF-α inhibitors are generally well tolerated among patients, increasing cases associated with psoriatic lesions after starting TNF-α inhibitor treatment have been reported. Based on our analysis, this sequela is most common in female patients and those using infliximab, which is most commonly associated with the onset of plaque psoriasis, as opposed to other psoriatic morphologies. Topical corticosteroids with discontinuation of TNF-α inhibitors may control or remit the psoriatic lesions. These cases demonstrate the importance of close monitoring of patients on TNF-α inhibitors for early recognition and treatment. Further research may provide a more detailed underlying immunological mechanism of paradoxical psoriasis and help target those individuals’ pre-initiation of TNF-inhibition who may have a predilection to develop paradoxical psoriasis.


Finerenone is a novel non-steroidal mineralocorticoid receptor (MR) antagonist (MRA) with high binding affinity, high MR selectivity and a short plasma half-life. In two major endpoint-driven clinical trials in patients with chronic kidney disease and type 2 diabetes mellitus (FIDELIO-DKD and FIGARO-DKD), finerenone induced significant cardiorenal protective actions, and has been recently approved for treatment of these patients. Heart failure with preserved ejection fraction (HFpEF) is a devastating clinical syndrome with increasing prevalence and poor prognosis. Pharmacological therapy of HFpEF is very limited and new therapeutic options are urgently needed. Finerenone has been shown to improve multiple pathophysiological parameters of HFpEF in preclinical models. In consonance, pre-specified subgroup analyses of FIDELIO-DKD and FIGARO-DKD suggested a potential beneficial effect of finerenone in HFpEF. This review will discuss the pharmacodynamic and -kinetic profile of finerenone. We will provide a general overview over the complex pathophysiology of HFpEF and data from pre-clinical studies, focusing on how finerenone improves multiple components of this pathophysiology. Finally, we will discuss current and future clinical trials with finerenone in heart failure patients focusing on HFpEF. The mineralocorticoid receptor (MR) belongs to the family of nuclear hormone receptors . PharmacRecently, a new class of MRA has been developed, named non-steroidal MRA, among which finerenone is the one with the most advanced global clinical development program . In two HFpEF is a clinical syndrome accounting for over 50% of the estimated 64.3 million heart failure patients worldwide . The 5-yIn summary, this review will discuss the pharmacology of the novel non-steroidal MRA, finerenone, and its potential use for the treatment of patients with HFpEF.50: spironolactone: 24nM and eplerenone: 990nM)  [Nowadays, two steroidal MRA are in routine clinical use, spironolactone and eplerenone , 20, 21.MPHASIS) . RegardiMPHASIS) .In summary, there is compelling evidence for the use of steroidal MRA in HFrEF to improve morbidity and mortality in these patients. However, this significant efficacy is associated with an increased risk of developing hyperkalemia, which requires close monitoring. In contrast, the evidence in HFpEF for MRA use is currently much less convincing and is discussed below.To further improve the benefit-risk profile of MR-based therapy, new compounds without a steroidal molecule backbone have been recently developed and named non-steroidal MRA . Their dIn differentiation from steroidal MRA, most of these compounds appear to have a different binding mode to the MR-ligand binding domain (LBD), thereby inducing distinct pharmacodynamic actions . This ne≥ 20 to ≤ 60ml/min/1.73m2) will be treated with different doses of AZD9977 in combination with the SGLT2-inhibitor dapaglifozin (10 mg) or dapaglifozin (10 mg) alone (NCT04595370). The estimated enrollment will be 500 patients and the estimated study completion date is in December 2023 . Focusing on safety issues with novel non-steroidal MRA, most of the compounds induce hyperkalemia when compared to placebo, however, when compared to steroidal MRA this occurs to a lesser extent [With respect to their clinical efficacy esaxeronone, approved for the treatment of arterial hypertension in Japan, and KBP-5074 induce significant antihypertensive actions ranging between − 11.5mmHg systolic blood pressure (SBP) lowering for low doses and − 16.5mmHg for high doses , 38. SigFinerenone is a novel non-steroidal MRA with high binding affinity IC50: 18nM) and high MR selectivity over sex steroid hormone receptors, thereby lacking anti-androgenic and progestational side effects (Table 8nM and h≥ 25 to < 60mL/min/1.73m2 or UACR ≥ 300 mg/g and eGFR ≥ 25 to < 75 mL/min/1.73m2) and T2DM were randomized to placebo or finerenone (10 or 20 mg/d) [≥ 40% from baseline or renal death) compared to placebo  [After completion of a successful phase I/II clinical development program, two phase III clinical trials with finerenone were conducted in patients with T2DM and CKD. In FIDELIO-DKD, 5.734 patients with CKD (UACR 30 to < 300 mg/g and eGFR ≤ 90mL/min/1.73m2 or UACR ≥ 300 mg/g and eGFR ≥ 60mL/min/1.73m2) and T2DM [≥ 40% from baseline or renal death) occurred in 350 patients (9.5%) in the finerenone group and in 395 (10.8%) in the placebo group  [The second phase III trial, FIGARO-DKD was conducted in a similar patient population as FIDELIO-DKD and focusses with its primary CV-composite endpoint  on CV morbidity and mortality , 7. 7.43and T2DM , 7. The and T2DM , 7. All and T2DM , 7. Patiand T2DM , 7. Afteand T2DM , 7. The Drug-drug interactions with finerenone have been investigated in healthy male volunteers . FinerenRecent publications confirmed the protective actions of finerenone in cardiorenal disease. A large network meta-analysis showed that in patients with T2DM and CKD, finerenone led to a risk reduction in MACE, renal outcomes and hospitalization for heart failure . Certainolder vascular aging phenotype, a metabolic obese phenotype and relatively younger natriuretic peptide (NP) deficient phenotype [older vascular aging phenotype in 30–50%, the metabolic obese in 25–30% and the younger NP deficient phenotype in 15–35% of HFpEF patients [older vascular aging phenotype appears to be driven by noncardiac factors, such as vascular stiffness or CKD-associated renal factors in combination with cardiac factors precipitating the development of cardiac hypertrophy, left atrial myopathy, and right ventricular dysfunction[metabolic obese phenotype is provoked by noncardiac factors, such as systemic fat accumulation during obesity and/or impaired insulin/ glucose metabolism, as well as by direct alterations of cardiac metabolism [younger NP deficient phenotype is characterized by reduced NP production and increased NP clearance and might be driven by perturbations in the cell – matrix compartment since increased levels of syndecan-4 and matrix metalloproteinase (MMP)-9 have been detected [HFpEF is a clinical syndrome that now accounts for almost half of all heart failure cases with an increasing prevalence . The pathenotype . These otabolism , 50. Furtabolism . FinallyWhen discussing the pathophysiology of HFpEF, the importance of arterial hypertension should be emphasized. The prevalence of hypertension among patients with HFpEF ranges between 28% and 90% –55. In TA central pathophysiological pathway that probably overlaps in all phenogroups is the inflammatory – fibrotic pathway . A subclCurrent drug therapy for HFpEF is very limited. SGLT-2 inhibitors are recommended as first-line therapy since they improve prognosis . Two ranIn summary, new therapeutic approaches to improve the prognosis of HFpEF are imperative and based on our current pathophysiological knowledge and post-hoc analysis of major event-driven clinical trials, we will discuss the potential benefit of MR-blocking drugs in this context.Steroidal MRA are first-line therapy in HFrEF together with ACE-I or ARNI, beta-blocker and SGLT2-inhibitors . Their r≥ 45%) in the TOPCAT trial [Spironolactone was investigated in 3.445 HFpEF patients  and diastolic dysfunction randomized to spironolactone or placebo [In addition to TOPCAT, spironolactone has been tested in the Aldo-DHF (Aldosterone Receptor Blockade in Diastolic Heart Failure) trial which included 422 ambulatory patients with HFpEF  trial . An incr2FPEF score, to these models [Finerenone is a novel non-steroidal, highly selective MRA with high binding affinity. It has been tested in various rodent models related to HFpEF. To draw conclusions from results of pre-clinical studies in HFpEF for clinical translation, strict criteria must be applied to the phenotyping of these HFpEF rodent models. Recently, Withaar and colleagues analyzed various rodent HFpEF models for their relevance to clinical HFpEF symptoms, clinical signs, and diagnostic criteria by applying two established clinical algorithms, the HFA-PEFF and He models . They foe models . HoweverAs described in detail above, inflammatory and fibrotic processes play a central pathophysiological role in the development of HFpEF. For Finerenone, significant anti-inflammatory and antifibrotic effects have been demonstrated in multiple pre-clinical studies. For instance, finereone reduced the cardiac accumulation of macrophages (CD68-positive cells) in a model of cardiac dysfunction induced by short-term isoproterenol application . Potent 2FpEF score, is the presence of diastolic dysfunction [Cardiac hypertrophy is a key criterion for HFpEF in the HFA-PEFF score . Finerenfunction , 80. Usifunction , 75, 81.function , 82. Finfunction , 76. Thefunction , 76, 81.2FPEF), finerenone exhibited beneficial actions on several of these parameters in preclinical studies including on cardiac hypertrophy, on diastolic dysfunction, on early alterations of systolic LV-function and on blood pressure regulation  . In one Two recent prespecified subgroup analyses of the FIDELIO-DKD and FIGARO-DKD studies investigated the efficacy of finerenone in patients with CKD and T2DM with and without heart failure , 85. Pat≥ 40%) (NCT04435626) / 115 mg/m2 , septal thickness or posterior wall thickness ≥ 1.1 cm and increased blood levels of NT-prBNP will be included [≤ 60mL/min/1.73m2), 40 mg finerenone (eGFR > 60mL/min/1.73m2) or placebo [Taken together, both the preclinical data and the currently available clinical data with finerenone demonstrate a yet to be precisely defined therapeutic potential of this non-steroidal MRA in patients with HFpEF. Finally, this question will be answered in the ongoing FINEARTS-HF Study to Evaluate the Efficacy and Safety of Finerenone on Morbidity & Mortality in Participants with Heart Failure and LV-Ejection Fraction NCT0443566 , finerenone induces significant cardiorenal protective actions. Beyond these results, finerenone has shown to improve multiple pathophysiological parameters of HFpEF in preclinical models. In consonance, pre-specified subgroup analyses of FIDELIO-DKD and FIGARO-DKD suggested a potential beneficial effect of finerenone in HFpEF.HFpEF is a devastating clinical syndrome with increasing prevalence and poor prognosis. Unfortunately, current guideline-recommend drug therapy in HFpEF is very limited with only one prognosis-improving drug class, SGLT2-inhibitors. Against this background, the potential beneficial effects of finerenone in HFpEF appear very promising. The final efficacy/ risk profile of finerenone in HFpEF is currently being investigated in over 6.000 patients in the FINEARTS-HF study, the results of which are desperately awaited to allow this drug to be used in the treatment of HFpEF.


Despite emerging evidence suggesting the efficacy of psilocybin in the treatment of mood disorders such as depression, the exact mechanisms by which psilocybin is able to elicit these antidepressant effects remains unknown.As the use of psilocybin as a treatment modality for depression has garnered increasing interest, this study aims to summarize the existing evidence of the mechanism of action with which psilocybin alleviates depressive symptoms, focusing specifically on the neurobiological effects of psilocybin in human subjects.Four databases  were searched using a combination of MeSH terms and free text keywords in September 2021. The original search included both human and animal studies and must have included testing of the mechanism of action of psilocybin. Only antidepressant effects were considered, with no other mood disorders or psychiatric diagnoses included. Two independent researchers screened at every stage of the review, with a third researcher resolving any conflicts. Though a full systematic review outlining the current literature on the complete mechanisms of action of psilocybin on depression was conducted, this abstract will focus specifically on the nine papers that included human subjects, disregarding the five animal models. PROSPERO registration number: 282710.After removing duplicates, the search identified 2193 papers and forty-nine were selected for full text review. Out of nine papers outlining the mechanisms of action of psilocybin use in human subjects, three papers investigated psilocybin’s effect on serotonin or glutamate receptor activity, two found an increase in synaptogenesis in regions such as the medial frontal cortex and hippocampus. Four found variation in blood flow to the amygdala, two found altered blood flow to the prefrontal cortex, and one found a reduction in delta power during sleep. Four papers found changes in functional connectivity or neurotransmission, most commonly in the hippocampus or prefrontal cortex.Overall, the exact mechanism of psilocybin’s potential antidepressant effect remains unclear. Multiple pathways may be involved, including alterations in serotonin and glutamate receptor activity, as well as shifts in amygdala activity, neurogenesis, and functional connectivity in various brain regions. The relative lack of studies, and the variety of neurobiological modalities and endpoints used challenged the consolidation of data into consensus findings. Further studies are needed to better characterize psilocybin’s mechanism of action and to better understand the clinical effects of the use of psilocybin in the treatment of depression.None Declared


We present the complete genome sequence of two Actinobacteriophages, OmniCritical and Barkley26, isolated in Clark County, NV. Over two semesters, The University of Nevada, Las Vegas (UNLV) students isolated and purified phages and manually annotated the genomes. The courses follow the HHMI Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Sciences (SEA-PHAGES) curricula. Hypoestes phyllostachya) grown at UNLV . Phage isolation and purification followed the HHMI Phage Discovery Guide protocols with Mycobacterium smegmatis mc2 155 as the host (10–1011) lysates were generated for DNA isolation and transmission electron microscopy (TEM). Genomic DNA was isolated using a commercial kit (Norgen Biotek), and both DNA samples were sequenced with the Illumina MiSeq System. The total reads for OmniCritical (SRX14483223) and Barkley26 (SRX14443484) were 777,403 and 737,822, respectively. Newbler  was used for de novo assembly of the raw reads, generating a single contig as described in reference  and Barkley26 from the soil of a polka dot plant , CD-Search , and HHPred ; Aragorn was used to identify tRNA genes  and Phage Commander, which summarize query results from Glimmer (v3.02b), Genemark (v2.5), Genemark Hmm (v2.0), GenemarkS (v4.28), GenemarkS2, Genemark Heuristic (v.2.0), Prodigal (v2.6.3), RAST (v2.0), and Metagene (v2.0) 8.8 and P13. PutatNA genes –18. DefaA genes – using thA genes –. OmniCriublished –24. Intehttps://phagesdb.org).OmniCritical and Barkley26 are temperate Siphoviruses that produced 4.0 and 5.0 mm diameter plaques, respectively. The capsid diameter and tail length of OmniCritical are 79.1 and 232.6 nm; for Barkley26, these dimensions are 58.6 and 245.3 nm . OmniCri


SLC11A1 gene impact risk of developing infectious, inflammatory, and endocrine disorders. This study is aimed to investigate the association between the SLC11A1 gene polymorphisms  and anthropometric and biochemical parameters describing T2DM. Eight hundred participants (400 in each case and control group) were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and amplification-refractory mutation system-PCR (ARMS-PCR) methods. Lipid profile, fasting blood sugar (FBS), hemoglobin A1c level, and anthropometric indices were also recorded for each subject. Findings revealed that SLC11A1–rs3731864 G/A, –rs17235416 (+ TGTG/− TGTG) were associated with T2DM susceptibility, providing protection against the disease. In contrast, SLC11A1–rs3731865 G/C conferred an increased risk of T2DM. We also noticed a significant association between SLC11A1–rs3731864 G/A and triglyceride levels in patients with T2DM. In silico evaluations demonstrated that the SLC11A2 and ATP7A proteins also interact directly with the SLC11A1 protein in Homo sapiens. In addition, allelic substitutions for both intronic variants disrupt or create binding sites for splicing factors and serve a functional effect. Overall, our findings highlighted the role of SLC11A1 gene variations might have positive (rs3731865 G/C) or negative (rs3731864 G/A and rs17235416 + TGTG/− TGTG) associations with a predisposition to T2DM.Diabetes, a leading cause of death globally, has different types, with Type 2 Diabetes Mellitus (T2DM) being the most prevalent one. It has been established that variations in the  Type 2 DM (T2DM) is a chronic disease whose global prevalence has reached worrying levels. In 2019, 463 million adults had T2DM, showing a three-fold increase worldwide compared to 20 years before the report, according to the International Diabetes Federation2. T2DM has a complicated etiology and is impacted by a broad spectrum of risk drivers, some of which are inevitable (such as age and genetic diversity) and others controllable 3. Dysregulation of metabolisms of proteins, lipids, carbohydrates, and nucleic acids might cause metabolic diseases through hereditary or environmental factors. Nonalcoholic fatty liver disease (NAFLD) is also caused by excessive body fat and insulin resistance, the two most important risk factors for type 2 diabetes. Overeating, a poor diet, and a stationary lifestyle are other contributors leading to T2DM, especially in persons with genetic predispositions4.Diabetes mellitus (DM) is a high prevailing and rapidly developing chronic endocrinological illness characterized by abnormal blood glucose levels3. Although most of the prior investigations on T2DM were conducted in Western or European societies, it is clear that the ways the disease develops are diverse in racial groups, proposing that a one-size-fits-all perspective might not be the best when determining risk drivers. It has been acknowledged that mainstream models pursued globally, such as the Western dietary pattern and endangered environmental sustainability, raise the risk of T2DM and associated comorbidities5. These factors and other socioeconomic and cultural impacts are attributed to the rise in overweight, a well-known element for T2DM6. Genome-wide association studies (GWAS) and Mendelian randomization (MR) demonstrates that genetic polymorphisms are risk factors for human diseases7. Over 140 gene loci have currently been linked to T2DM by GWAS and other sequencing studies. Fifteen of these loci encode membrane transport proteins that are either known or hypothetical8. T2DM is a polygenic disorder that is influenced by more than 400 genetic variations, according to extensive GWAS9. There appears to be only a little predictive value in these variations over other conventional contributors, such as corpulence, a sedentary lifestyle, and poor diets in T2DM development11. Furthermore, though discrepancies in the distribution and prevalence of T2DM risk alleles have been found among races, there is little proof to support the idea that these variants account for racial dissimilarities in T2DM predisposition1. A growing emphasis is on how gene-environment interactions occurring during in utero development might affect the risk of developing cardio-metabolic disorders in adulthood; that is why assessing the interaction of behavior and genetics is essential. The developmental origins of the health and disease (DOHaD) framework, which has been linked to T2DM development and other non-communicable illnesses, are consistent with the current research12.The large consumption of red and processed meats, refined carbohydrates, and simple sugars defines the Western dietary pattern. This pattern has gained popularity worldwide and has been strongly related to an elevated risk of T2DM13. The Slc5, Slc13, Slc16, Slc25, and Slc30 gene families investigated in different tissues and organs, such as the pancreas, liver, gut, adrenal glands, skeletal muscle, and fat, and have been associated with metabolic diseases such as overweight, NAFLD, and T2DM in both human and ratty research4. It has been established that metformin's bioavailability, clearance, and pharmacological effect in T2DM are significantly influenced by the expression of the solute carrier proteins Slc22A1, Slc22A2, Slc22A3, and Slc47A115. There has yet been no mention of a thorough analysis of SLC genes in obesity16.Nutritional components and environmental chemicals interact with genes to maintain regular activity in the body's complicated health system. Numerous research has been conducted on the essential nutrients and metabolites, but the mechanisms by which they are transported inside the body have received very little attention. Membrane transporters predominantly consist of ATP-binding cassettes (ABCs) and solute carrier (SLC) transporters, which are members of the ion and water channelsSLC11A1 [formerly known as natural resistance-associated macrophage protein 1 (NRAMP1)] is abundantly expressed in monocytes and phagocytes17. The gene encoding SLC11A1 in humans is 14 kb in size and contains 15 exons. This gene clusters with other genes in close proximity to its location on chromosome 2q35, in a region of high linkage disequilibrium (LD) that spans about 400 kb18. SLC11A1 has been shown to control the susceptibility to Salmonella, Mycobacterium, and Leishmania infections within cells19. SLC11A1's role in the discharge of Fe2+, Mn2+, and Co2+ from phagosomes may prevent vacuolar pathogens' access to these essential micronutrients20. According to a study by Yang et al. the SLC11A1–rs3731685 G/A variation might correlate with Type 1 DM (T1DM) risk in a large-scale survey of 8463 cases and 9835 controls18.In humans, the host resistance factor SLC11A1 variations known to alter SLC11A1 transcription and functioning is the INT4 polymorphism (469 + 14G/C or rs3731865), which is positioned in exon 4a22. Furthermore, the 3'UTR (1729 + 55del4 or rs17235416) variation refers to a 4 bp insertion/deletion immediately 3' of the stop codon24; nevertheless, the potential role of this polymorphism on the functioning and expression of SLC11A1 has not yet been established. With this background, the present study aimed to unveil the possible correlation between SLC11A1 variants, including rs3731865G/C, rs3731864 (577-18G/A) in intron 5, and rs17235416 + TGTG/−TGTG polymorphisms and the risk of T2DM in an Iranian population. Figure One of the 25. The diagnosis was made according to the 2018 American Diabetes Association (ADA) Standards of Medical Care in Diabetes. The healthy, non-diabetic control group was selected from those with FBS levels < 99 mg/dL and HbA1c levels < 5.7% (to weed out pre-diabetic ones). Individuals with late-stage autoimmune or malignant diseases, gestational diabetes, polycystic ovary syndrome, metabolic syndrome, chronic renal failure, hypertension, and pregnant women were excluded from the study. The human-involved procedure was under the 1964 Helsinki declaration, and the ethics committee of Zahedan University of Medical Sciences approved the study's protocols . The webpage of the ethics certificate is available at https://ethics.research.ac.ir/EthicsProposalViewEn.php?id=189533. Before enrollment, informed consent was obtained from all subjects or their legal guardians.Eight hundred participants (400 in each case and control group) were selected among individuals referred to the Diabetes Clinic of Bu-Ali Hospital, Zahedan, Iran. The case group was of individuals with T2DM who had fasting blood sugar (FBS) levels of ≥ 126 mg/dL and hemoglobin A1c (HbA1c) levels of ≥ 6.5%For each participant, a total of 5 mL of whole blood was drained in ethylenediaminetetraacetic acid (EDTA)-containing- or serum clot activator tubes. The former tube was used for DNA extraction and HbA1c measurement, and the latter was utilized for measuring FBS and lipid indices  using commercial spectrophotometric kits .https://clincalc.com/stats/SampleSize.aspx). The server uses the below formula to calculate sample size.To calculate sample size, we conducted a pilot study to collect blood samples from a small population  and genotyped all of the examined SNPs. This allowed us to identify an adequate sample size. The chi-square test was then used to calculate the allelic frequencies of the investigated variants in both groups. The estimated frequencies were then subjected to a sample size analysis utilizing the sample size calculator server's online version  with an inelastic measuring tape to within 0.1 cm. The widest hip area and its maximum bulge were determined to measure the hip circumference with an accuracy of 0.1 cm. Waist-to-hip ratio (WHR) was calculated by dividing the waist circumference by the hip circumference in centimeters. The conicity index (CI) was calculated as previously described by Shidfar et al.27. The purity and concentration of extracted DNA were determined by calculating the 260/280 optical density ratio using a Nanodrop device . Data for selecting variations and designing specific primers were acquired from National Center for Biotechnology Information (NCBI) database. Specific primers were designed using the Gene Runner® v.6.5.52 Beta software and produced by GenFanAvaran Company in Iran.Genomic DNA was extracted from nucleated white blood cells using a simple salting-out techniqueSLC11A1–rs3731864 and –rs3731865 SNPs) and amplification-refractory mutation system-PCR (ARMS-PCR) (for SLC11A1–rs17235416) techniques. The reaction mixture had a final volume of 20 μL and contained 0.9 μL of genomic DNA (~ 60 ng/mL), 0.8 μL of each primer (8 pmol), 10 μL of 2 × Taq PreMix , and 7.5 μL of double-distilled water. The mixture was cycled using a Techne thermal cycler  under the following conditions: initial denaturation at 95 °C for 5 min, 35 cycles at 94 °C for 30 s, specific annealing temperatures (based on Supplementary Table Studied variations were genotyped by applying the polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) (for MspI (for SLC11A1–rs3731864 G/A) or ApaI (for SLC11A1–rs3731865 G/C) restriction enzymes  and incubated for 10 h at 37 °C. PCR products were then electrophoresed on 1.5% agarose gel stained by GreenViewer dye . DNA bands were photographed under ultraviolet  was recruited for data analysis. Deviation from Hardy–Weinberg Equilibrium (HWE) was assessed via Pearson's Chi-square test. Continuous variables were compared between cases and controls using standard single sample t statistic, Mann–Whitney–Wilcoxon, and Pearson Chi-Square tests where appropriate and expressed as mean ± standard deviation (SD). Odds ratio (OR) and 95% confidence intervals were calculated to estimate the relative risk of the disease. Binary logistic regression analysis was employed to examine the correlation between the clinical-demographic findings of the studied groups and T2DM risk. Besides, haplotype analysis was conducted through the online SHEsis softwareSLC11A1 on the pattern of splicing processes29. SpliceAid is a web-based tool collecting all the experimentally assessed target RNA sequences that are bound by splicing proteins in humans. Using the SpliceAid server, the user submits sequences, and the server identifies the exact correspondence between the sequences submitted and the sequences in the database, giving accurate and dynamic graphic results.A complex set of RNA-binding proteins controls the post-transcriptional processing of RNA, such as capping, polyadenylation, splicing, export, and the protein's secondary structure. Allelic substitution in DNA can affect some of these processes, primarily the accuracy and efficiency of splicing, by altering the complex of proteins bound to the pre-mRNAs. Knowing the RNA sequences recognized by each protein involved in post-transcriptional RNA processing is necessary for predicting the effects of mutations at both RNA and protein levels. For this purpose, we recruited the SpliceAid database to determine the impact of studying both intronic variants of WebLogo v.2.8.2 server was employed to identify the preserved regions of all three studied polymorphisms30. Using WebLogo, sequence logos are generated, representing patterns within multiple sequence alignments. In comparison with consensus sequences, sequence logos provide a more detailed and more accurate description of sequence similarity and can be used to quickly reveal characteristics of an alignment that would otherwise be difficult to detect. An individual logo consists of a stack of letters at each position in the sequence, one stack for each letter. Stack heights (measured in bits) represent sequence conservation at each position, and symbol heights reflect the relative frequency of amino acids or nucleic acids at each position30. As defined by Schneider and Stephens, sequence conservation is the difference between the entropy of the observed symbol distribution and the maximum possible entropy31. In particular, sequence logos offer a richer and more detailed description of, for example, a binding site, as compared with consensus sequences32. Sequence information of genomic DNA in different formats, including CLUSTALW, FASTA, MSF, NBRF, PIR, NEXUS, PHYLIP, and plain flat-file, can be entered into the Weblogo server (available at https://weblogo.threeplusone.com/create.cgi) for multiple sequence alignments. Depending on how frequently they occur, various SNPs are scaled.The https://inbio-discover.intomics.com/map.html) was employed33. The database is a comprehensive and accurate PPI resource built from more than six million traceable entries, showing a set of highly trusted interactions between proteins based on experimentally determined databases. This research put the network expansion in the “Include neighboring proteins” mode to show close and related proteins in terms of expression, regulation, or function. In this connection, pathway interactions are indicated as lines, and the remainder is inBio Map™ high-confidence interactions. Data were generated by entering the UniProt ID for the SLC11A1 protein, P49279, into the server. UniProt is an online reservoir for proteins that extracts data from the Swiss-Prot, TrEMBL, and PIR-PSD databases34. Expression, regulatory, and function-related proteins were made available through the network expansion method of this database. In order to design an interaction network for SLC11A1 as a hub gene, information regarding the known and/or predicted interactions, gene fusion, co-expression, and protein homology was obtained using STRING34. STRING imports protein association knowledge from databases of physical interactions and databases of curated biological pathway knowledge . Finally, inBio Discover™ was utilized to analyze the PPI network.To predict the protein–protein interaction (PPI) network of the SLC11A1 protein, the newest version (01-04-2022) of the web-based inBio Discover™ database . At the same time, FBS, TG, TC, HDL-c, LDL-c, conicity index, and body mass index (BMI) were significantly different between cases and controls (p < 0.001).The case group consists of 400 patients with T2DM  and 400 healthy control subjects . As shown in Table SLC11A1 gene variants in controls and T2DM cases. None of the studied variations deviated from HWE in cases or controls . We found a strong link between the rs3731864 G/A variant and T2DM under codominant1 GA vs. GG , dominant GA + AA vs. GG , and overdominant GA vs. GG + AA  genetic patterns. Moreover, the A allele of rs3731864 G/A decreased T2DM risk by 54%. Similarly, The rs17235416 variant was associated with a decrease in T2DM risk under codominant1 Ins/Del vs. Ins/Ins , dominant Ins/Del + Del /Del vs. Ins/Ins , and over-dominant Ins/Del vs. Ins/Ins + Del /Del  modes of inheritance. Deletion of the TGTG repeat in this polymorphism conferred protection against T2DM . In contrast, compared with the healthy controls, T2DM risk was dramatically increased in patients carrying the CG , CG + GG  genotypes of rs3731865 G/C. Furthermore, an increase in T2DM risk was found under the allelic (G vs. C) as well as the overdominant (CG vs. CC + GG) model of this single nucleotide variation (SNP) .Table SLC11A1 SNPs and laboratory findings and the demographical characteristics of the studied groups are shown in Table SLC11A1–rs3731864 G/A and TG levels in patients with T2DM (p = 0.048). The SLC11A1–rs3731865 C/G variant was associated with WC and LDL-c levels of the healthy controls . Moreover, the SLC11A1–rs17235416 Ins /Del variant was associated with WC, conicity index, and HDL-c levels in controls .The correlation between the SLC11A1–rs3731864 G/A, –rs3731865 G/C, and –rs17235416 +TGTG/−TGTG haplotypes in T2DM cases and healthy controls. We found a higher frequency of the SLC11A1–rs3731864 G/A, –rs3731865 G/C, and –rs17235416 +TGTG/−TGTG haplotypes in patients with T2DM compared with controls. Compared with the reference haplotype (G/C/ + TGTG), the A/C/ + TGTG haplotype of rs3731864/rs373186/rs17235416 significantly diminished T2DM risk in our population . The linkage disequilibrium (LD) between three SLC11A1 polymorphisms was also calculated, and no strong LD was found between the three studied variations , the genotype combination of GA/CC/Ins-Ins markedly increased T2DM risk by 1.66 folds , whereas the GA/CC/ Ins-Ins combination diminished T2DM risk by 57% .Table WebLogo server demonstrated that all three studied polymorphisms, especially SLC11A1–rs3731864 G/A and –rs3731865 G/C, resided in unconserved regions across multiple mammalian species  and ATPase copper transporting alpha (ATP7A) proteins have direct interactions with the SLC11A1 protein in Homo Sapiens. According to the known interactions , the ATP7A in and of itself interacts with SLC11A2, solute carrier family 31 member 2 (SLC31A2), and antioxidant 1 copper chaperone (ATOX1) in Homo sapiens 38 of some genes have been found to independently contribute to the predisposition to T2DM, suggesting that there may be other, as-yet-undiscoverable functional variations in Homo sapiens. Additional in-depth population genetic research will be required to comprehend the connection between more complicated haplotypes and disease development in various geographical areas39. In the present work, for the first time, we sought to investigate the correlation between SLC11A1 variants and the risk of T2DM in a sample of the Iranian population. Our findings showed a significant association between SLC11A1 polymorphisms and T2DM risk, where rs3731865 G/C markedly enhanced T2DM risk and both rs3731864 G/A and rs17235416 + TGTG/− TGTG variants significantly diminished the risk of this endocrine diseases under different genetic models.In the last 30 years, the prevalence of T2DM has doubled, making it one of the most significant global health issuesSLC11A1, most previous reports focused on the role of SLC11A1 variants in the pathogenesis of autoimmune and infectious diseases40, such as HIV41. It was also suggested that the (GT)n polymorphism's high-expressing allele (iii) and low-expressing allele (ii), respectively, might be responsible for susceptibility to these conditions. This has also been confirmed in numerous studies on autoimmune and infectious diseases, including tuberculosis, demonstrating that there may be some balance in selecting factors that maintain both alleles in the population39. In another study by Ling et al. (2014), it was reported that the A allele in SLC6A20–rs13062383 increases the susceptibility to T2DM in populations with different genetic backgrounds42. Xu et al. concluded that T2DM is associated with the AA genotype of SLC30A8–rs11558471 in Homo sapiens. Haplotype A/C/A seems to be a risk factor, and haplotype A/C/G may be a protective factor against T2DM in the Han population43. The results of Chen et al.'s meta-analysis in 2015 showed that SLC30A8 rs13266634 might be a crucial genetic contributor to the risk of T2DM among Asians and Europeans but not Africans. It also indicated that people with the CC genotype have a 33.0% and 16.5% higher risk of T2DM compared with those with TT and CT genotypes, respectively14. According to research by Zaahl et al., the promoter SNP rs7573065 (− 237 C/T) plays a protective role in the contribution of SLC11A1 to inflammatory bowel disease44. They also revealed that when allele 3 of the 5′ microsatellite was present, the allele C to T alteration at the position of 237 (rs7573065) downregulated SLC11A1 to a level comparable to that observed allele 2 of the microsatellite45.In terms of SLC11A1 downregulation in NOD mice mimicked the protective Idd5.2 T1DM-resistant haplotype and decreased the prevalence of T1DM46. It was found that this gene affects the ability of dendritic cells (DCs) to process and present pancreatic islet antigens , increasing the stimulation of a diabetogenic T-cell clone47. Unfortunately, there is a lack of evidence for the involvement of SLC11A1 variants in the etiology of DM. In this regard, Yang and colleagues concluded that the SLC11A1 gene variant rs3731685 (INT4) might be correlated with T1DM risk in a population of European ancestry. Although they found no correlation between mRNA levels of SLC11A1 and different genotypes of this SNP in whole blood samples, a possible association with purified cell subsets, particularly monocytes or macrophages, could not be completely ruled out48. In another cohort study, Takahashi et al. examined the SNPs located in the promoter region of SLC11A1, which might affect transcriptional activity, in 224 controls and 95 Japanese cases of T1DM. Japanese participants have been found to carry the specified alleles 2, 3, and 7. They found a significant difference in the subset of Japanese individuals with T1DM; these patients had considerably higher allele 7 frequencies than the healthy subjects, as did those carrying no susceptibility HLA class II haplotypes, DR4-DQ4 or DR9-DQ9. Overall, they concluded that the new promoter variant of SLC11A1 impacts Japanese individuals' susceptibility to T1DM49. Mycobacterium avium subsp. paratuberculosis (MAP) has been linked to the onset of T1DM; accordingly, Paccagnini et al. examined 59 T1DM cases and 79 healthy individuals for 9 SLC11A1 SNPs and the presence of MAP using the PCR technique. Blood levels of MAP DNA and the 274C/T SCL11A1 polymorphism were discovered to be linked to T1DM. Because MAP is not degraded by macrophages and is processed by DCs, it is important to determine whether mutant variants of SLC11A1 affect the processing or presentation of MAP antigens, which could lead to an autoimmune disorder and T1DM50. In agreement with these reports, we found a negative association between T2DM and two SNPs in SLC11A1 (rs3731864 G/A and rs17235416) and a positive association between the disease and SLC11A1 rs3731865 G/C.Kissler et al. discovered that SLC11A1 gene. Interestingly, results of our web-based analysis showed that the A allele of SLC11A1 rs3731864 disrupts the binding sites of SC35, SF2/ASF, heterogeneous nuclear RNA protein (hnRNP) F, hnRNP H3, hnRNP H1, and hnRNP H2, whereas the minor allele of SLC11A1 rs3731865 creates the binding site for SF2/ASF and hnRNP H3, as splicing factors. This is important because splicing factors are involved in regulating distinct gene expression processes51. It has been documented that alternative splicing via SF2/ASF52, hnRNP F54 can contribute to the pathogenesis of diabetes or cause insulin resistance. Furthermore, overexpression of the hnRNP H1 was also observed in the nucleus of Inflamed Islets of fulminant T1DM55. A distinct binding specificity has been reported between the human splicing factors ASF/SF2 and SC35, and these specificities are functionally important56.Both of the studied intronic variants were located in the splicing sites of the 57. Since proteins cannot act alone, most cellular functions depend on interactions between them. In the current study, we have utilized the inBio Discover™ to explore possible interactions between SLC11A1 and other proteins to gain valuable insight into a complex interaction network that may be responsible for the onset of T2DM. This is important since, to the best of our knowledge, the role of this solute carrier protein has not been studied in T2DM patients. Our Bioinformatics results showed that some of the genes that directly interact with SLC11A1 can also play important roles in the course of T2DM, making SLC11A1 a hub protein that can regulate different signaling pathways involved in the pathogenesis of T2DM. SLC11A1, a divalent cation transporter, plays an important role in early macrophage activation and exerts multiple pleiotropic effects on macrophage function, including on the expression of chemokines, IL-1β, tumor necrosis factor α (TNF-α)-inducible nitric oxide synthase, and MHC class II molecules. The multiple pleiotropic effects of SLC11A1 on macrophage function suggest that SLC11A1 is a prime candidate for T1DM in humans and mice49.An interaction network between proteins comprises a few highly connected nodes (known as hubs) and many poorly connected nodes. In genome-wide studies, it has been established that the deletion of a hub protein increases the likelihood of death, a phenomenon known as the centrality-lethality rule. A key notion of systems biology lies in the biological significance of network architectures, which are believed to reflect the special role hubs play in organizing networks58. Ferroptosis is also associated with diabetic cognitive dysfunction, and a previous study has shown that Slc40a1 mediates ferroptosis in T1DM59. Accordingly, we found that SLC11A1 and ATPase copper transporting alpha (ATP7A) are the most relevant proteins interacting with SLC11A1. This is important since it has been established that ATP7A60, fibrinogen chain beta (FGB)61, fibrinogen chain alpha (FGA)62, Solute carrier family 11 member 2 (SLC11A2)58, and Solute carrier family 40 member 1 (SLC40A1)63 might have essential roles in the pathogenesis of T2DM. Interestingly, hemostatic dysfunction and subclinical inflammation might play a role in the complex etiopathogenesis of diabetic peripheral neuropathy (DPN). Fibrinogen is involved in both hemostatic and inflammatory pathways, and it is hypothesized that fibrinogen gene polymorphisms might be associated with DPN64. In general, various studies have shown this gene to be related to the pathogenesis of T2DM and SLC11A1. These evidences suggest that SLC11A1 may act as a regulatory hub for controlling cell’s metabolism and activity through controlling the activity of other genes. Further investigation on the relationship between these proteins and the investigated receptor are warranted.The main mediator of iron transfer is SLC11A2, and iron is absorbed through this apical transporter in intestinal epithelial cells and macrophages. Previous studies have demonstrated that iron metabolism can affect insulin sensitivity, leading to T2DM30. From a clinical perspective, SNPs are potential diagnostic and therapeutic biomarkers for many types of cancer and metabolic disorders. Those in the promoter region affect gene expression by altering the promoter activity, binding of transcription factors, DNA methylation, and histone structure. Introns comprise approximately half of the human noncoding genome and have critical regulatory roles in gene regulation and expression. SNPs in intronic regions might cause diseases and alter the genotype–phenotype association by generating splice variants of transcripts and promoting or disrupting the binding and function of long noncoding RNAs (lncRNAs) (such as rs3731864 G/A and rs3731865 G/C). SNPs in the 5′-UTR regions can potentially affect translation, whereas those in the 3′-UTR region  impact the binding of microRNAs (miRNAs)66. Intronic sequences might be conserved, as they contain expression-regulating elements that impose functional constraints on their evolution68. SNPs located in these regions could be pathogenic even if they are conserved69. Variation analysis revealed that all three studied polymorphisms, specifically SLC11A1 rs3731864 G/A and rs3731865 G/C, reside in unconserved regions across multiple mammalian species. Understanding the mechanisms underlying the effects of SNPs that result in metabolic diseases such as diabetes is critical for elucidating their molecular pathogenesis.Perversions from the normal pattern represent DNA distortion or base flipping in sequenceSLC11A1 gene. The first and second variants are located in the regulatory regions; for example, splicing sites could potentially impact the expression of SLC11A1. Accordingly, the presence of a minor allele in these positions can affect post-transcriptional modifications and/or translation. Thus, it is hypothesized that nucleotide substitution in these locations is followed by producing a less or more efficient protein. However, the exact mechanism regarding the role of the SLC11A1-mutated protein is not understood yet and requires additional bioinformatics analyses.Based on the chromosomal position, rs3731864 is located 18 bp before exon 6, rs3731865 is located 14 bp after exon 4, and rs17235416 into exon 15 of the 70 and can thus be considered valuable prognostic markers for the disease. This is crucial because the lack of prevention and healthcare measures accounts for the fast rise in the prevalence of such endocrine disorders and their complications in developing countries. Iran lacks the most recent studies and knowledge necessary for effectively managing and treating T2DM. Our research aims to develop effective and affordable approaches to assess the genetic variation-based risk of T2DM development. In order to provide better treatment options against T2DM, we expect that our findings may help clinicians in the management and early detection of this condition. Additionally, creating a T2DM biobank for this cohort will be beneficial since this is the first study testing these SNPs in T2DM patients. Although SNPs in the SLC11A1 encoding gene's intronic and 3-UTR regions were chosen in the current study to study the relationship between SLC11A1 variations and the risk of T2DM, these variants might not provide a full picture of the SLC11A1 gene's genetic activity. As a result, a fine-mapping study may be needed subsequently. On the other hand, T2DM is a complicated metabolic disorder driven by environmental and genetic variables that were not examined in this study and can be considered a limitation. Furthermore, we have not performed Sanger sequencing to confirm our genotyping results, which can also be considered a limitation of the current study. Lastly, our sample size was relatively small, and this could potentially affect the outcome of such population-based studies. Despite these, we believe that the findings of our study highlight the essential role of SLC11A1 polymorphisms in predisposition to T2DM in subjects with Iranian ancestry.Accordingly, rare functional noncoding SNPs identified by large-scale whole genome sequencing have revealed unexplained heritability of T2DMSLC11A1 rs3731865 G/C is associated with an increased risk of T2DM in our population, while SLC11A1 rs3731864 G/A and rs17235416 + TGTG/−TGTG SNPs were correlated to decreased risk of developing this endocrine disease. Further bioinformatics analyses, along with replicated studies on different ethnicities, are needed to confirm our findings. Additionally, given the significant effect of these SNPs on the onset of T2DM, it appears likely that additional genetic variants in this gene may contribute to T2DM susceptibility. These findings may facilitate a detailed understanding of the molecular pathogenesis of T2DM and the genetic basis of heterogeneous susceptibility, with potential implications for the development of more effective therapeutic strategies.Our findings showed that Supplementary Information.


IntroductionHeart failure (HF) is a clinical syndrome with symptoms and/or signs caused by a structural and/or functional cardiac abnormality and corroborated by elevated natriuretic peptide levels and/or objective evidence of pulmonary or systemic congestion. Among HF types, HF with preserved ejection fraction (HFpEF) is the commonest form. However, the diagnosis and management of HFpEF are challenging. In addition, the perception of healthcare professionals (HCPs) towards the diagnosis and management of HFpEF patients differs due to the existing gap between the guidelines and daily clinical practice. Therefore, an online survey was conducted to understand the HCPs’ knowledge and practice gaps in the diagnosis, treatment, and management of patients with HFpEF.MethodsA total of 160 respondents, i.e., cardiologists, internists, and cardiology residents from different community-based practices and hospitals across Indonesia participated in an online continuing medical education (CME) survey. A questionnaire was formulated to assess awareness, current practice patterns, challenges, and confidence of the HCPs related to the HFpEF.ResultsHCPs stated that diagnosis of HF is the prime responsibility of cardiologists and general physicians but not of general internists. According to the HCPs, reduction in mortality, reduction in hospitalization, and improved quality of life are the most important goals of HF treatment. The perceived prevalence of HFpEF is estimated to be 30-60% and mortality rates of HFpEF and HF with reduced ejection fraction (HFrEF) are similar. Further, mixed types of responses with different combinations of diagnosis, treatment, and prevention, were obtained when HCPs were asked about the challenges faced in HFpEF. Among the therapies, angiotensin-converting enzyme (ACE) inhibitors, mineralocorticoid receptor antagonists (MRA), beta-blockers, and diuretics are frequently used for the treatment of HF.ConclusionThe perception of the HCPs toward the diagnosis and management of HFpEF may affect optimal care. Based on our findings, the cardiologists are well aware of the current situation of HF in Indonesia and treat patients with HFpEF effectively. Heart failure (HF) is a complex syndrome associated with increased mortality and morbidity. It is characterized by decreased functionality of heart muscles to pump blood, resulting in reduced quality of life. It has affected approximately 38 million people worldwide and 10 million people in Indonesia . StudiesIn 2020, the members of the Heart Failure Society of America (HFSA), the Heart Failure Association of the European Society of Cardiology (HFA/ESC), and the Japanese Heart Failure Society (JHFS) consensually defined HF as “a clinical syndrome with symptoms and/or signs caused by a structural and/or functional cardiac abnormality and corroborated by elevated natriuretic peptide levels and/or objective evidence of pulmonary or systemic congestion” . Based oFor effective diagnosis of the disease and management of patients with HF or particularly HFpEF, several guidelines, such as ESC and American Heart Association (AHA)/American College of Cardiology (ACC), have recommended various diagnostic tools to determine prognosis or disease severity and evidence-based approach, respectively. For instance, left ventricle (LV) structural or functional alterations, such as elevated natriuretic peptide levels, filling pressures, or hemodynamic measurements, may support the diagnosis of HFpEF. Further, the use of diuretics, sodium-glucose cotransporter 2 inhibitor (SGLT2i), angiotensin receptor-neprilysin inhibitor (ARNi), mineralocorticoid receptor antagonist (MRA), and angiotensin receptor blocker (ARB) are recommended therapies for the management of patients with HFpEF . HoweverBased on this background, the present study aimed to understand the perceptions of HCPs in terms of awareness, current practice patterns, challenges, and confidence related to the diagnosis, treatment, and management of patients with HFpEF, via an online survey to decipher the HCPs knowledge and practice gaps.An online continuing medical education (CME) survey was conducted to assess awareness, current practice patterns, challenges, and confidence of HCPs related to the diagnosis, treatment, and management of patients with HFpEF.Survey designing and developmentBased on the pertinent literature, ESC (2021) and ACC/AHA (2020) guidelines, and expertise in HF management, a questionnaire was formulated by a team of experts from the Indonesian Heart Failure Working Group. The Indonesian Heart Failure Working Group works under the Indonesian Heart Association (IHA) which aims to lower the National burden of heart failure and cardiometabolic disease . The surRespondentsA total of 160 respondents, i.e., cardiologists, internists, and cardiology residents from different community-based practices and hospitals across Indonesia participated in the survey. The respondent’s identity, practice location, and responses were kept anonymous and confidential. Additionally, the respondents were informed that answering the questionnaire would mean their consent to participate in the survey.Statistical analysesOnly completed surveys were included for statistical analysis. All data were analyzed using GraphPad version 8 . The Kolmogorov-Smirnov Goodness of Fit test was used to assess the normality of the data. Based on the data, the results are reported in frequencies and percentages. The data generated by multiple choice questions were analyzed using descriptive statistics.Demographic characteristicsIn total, 160 participants completed the survey. Among them, 87.5% (n=140) were cardiologists, 11.25% (n=18) were cardiology residents and two respondents were identified as internists. Of them, 55% (n=88) belonged to the age group of <40 years, 25% (n=40) were in the age range of 40-49 years, and the remaining 20% (n=32) respondents were 50 years or above. About 63.1% (n=101) of respondents were males and 36.9% (n=59) were females. Although HCPs from all the regions of Indonesia participated and most respondents were from Jakarta, Java, Bali, Sumatra, Banten, and Sulawesi. Notably, HCPs had been practicing for more than 10 years and treating >10 patients with HF per week. The demographic data of the respondents are detailed in Table Heart failure diagnosis and managementBased on the responses, 54.4% and 45.6% of respondents stated that the diagnosis of HF is the prime responsibility of cardiologists and general physicians, respectively. However, none of the respondents stated it as the responsibility of the general internist. When the respondents were asked to rank the goals of HF treatment, approximately 50% of respondents rated reduction in mortality as the most important goal followed by prevention of recurrent hospitalizations due to worsening heart failure, and then improvement in clinical status, functional capacity, and quality of life (QoL) Figure , panel aFurther, a list of HF medications, such as angiotensin-converting enzyme (ACE) inhibitors, ARB, beta-blockers, MRA, ivabradine, hydralazine/isosorbide dinitrate, SGLT2 inhibitors, ARNi, diuretics, and digoxin was given and asked to mark the essential, frequently used, and less frequently used medications. More than 75% of respondents stated that ACE inhibitors, ARNI, MRA, beta-blockers, SGLT2 inhibitors, and diuretics are essential medications for HF management. Among them, ACE inhibitors, MRA, beta-blockers, and diuretics are frequently used HF medications, as stated by >85% of respondents. Based on their responses, ivabradine, hydralazine/isosorbide dinitrate, SGLT2 inhibitors, ARNI, and digoxin are less frequently used HF medications. The data is shown in Figure HFpEF managementWhen HCPs were asked about the percentage of patients with HFpEF among their HF patients, 80/160 (50%) HCPs reported that 30-60% of patients were with HFpEF, 51/160 32%) HCPs reported the HFpEF patient percentage less than 30%, while the remaining 18% stated the percentage of greater than 60%. Regarding the awareness of HCPs on the mortality rate of HFpEF patients, 62% of respondents stated that the mortality rate among HFpEF patients is similar to HFrEF while 30% of respondents stated that only a few patients die due to HFpEF  ≥50%, objective evidence of cardiac structural and/or functional abnormalities, elevated natriuretic peptides, comorbidities, and objective evidence of high left ventricle filling pressure Figures , 4b.HF is a leading medical condition with substantially increasing prevalence in both developing and developed countries . Among tTo the best of our knowledge, this is the first Indonesian survey that assessed the HCPs’ knowledge and awareness concerning diagnosis, treatment, and management of patients with HF focusing HFpEF. In the present study, 99% (n=158) of the participants belonged to the cardiology department with a male predominance of 63%. More than half of the HCPs (55%) were <40 years of age. All the major regions of Indonesia were covered during the survey and the majority of the respondents were from Jakarta, Java, Bali, Sumatra, Banten, and Sulawesi. A similar study was conducted by Gupta et al. with 448 physicians from different regions of Canada .Regarding the diagnosis of HF, the participants responded that it is the prime responsibility of cardiologists and general physicians but not general internists. In a study, Hancock et al. conducted a national survey in England to assess changes in healthcare professionals’ views about the diagnosis and management of HF since 2003 . The autFurther, the respondents were asked to rank the goals of HF treatment, approximately 50% of respondents rated reduction in mortality as the most important goal followed by prevention of recurrent hospitalizations due to worsening heart failure, and then improvement in clinical status, functional capacity, and quality of life (QoL) Figure , panel aConcerning HFpEF, the knowledge and awareness of the HCPs were checked and they asked about the prevalence and mortality rate of HFpEF. About 50% of HCPs reported the prevalence of HFpEF at their clinic is between 30% and 60% which is in accordance with the data of Tromp et al., Mumpuni et al., and ESC guidelines 2022 . EvidencThen, physicians were asked about the difficulty in treating patients with HFpEF. Majority (79%) of the physicians agreed to find more difficulty in treating patients with HFpEF than patients with HFrEF. This is because patients with HFrEF can be readily diagnosed and the condition can be confirmed through cardiac imaging . HoweverIn a list of HF medications, the respondents were asked to mark the essential, frequently used, and less frequently used medications among ACE inhibitors (ACEi), beta-blockers, MRA, ivabradine, hydralazine/isosorbide dinitrate, SGLT2 inhibitors, ARNi, diuretics, and digoxin. Mixed responses were obtained. More than 75% of respondents marked ACEi, ARNi, MRA, beta-blockers, SGLT2 inhibitors, and diuretics as essential medications. Among these, ACE inhibitors, MRA, beta-blockers, and diuretics are frequently used and ivabradine, hydralazine/isosorbide dinitrate, SGLT2 inhibitors, ARNi, and digoxin are less frequently used HF medications. In a study, Reyes et al. reported that ACE inhibitors and diuretics are the major pharmacological treatments for HF in Indonesia . When thOur study has a few following limitations: (1) the sample size is very small for a large country like Indonesia. (2) Only two internists participated in the survey which limited the availability of comparative data of cardiologists and internists. (3) Mixed responses were obtained for many questions which could not be interpreted or discussed.Conclusively, several guideline-endorsed therapies exist to improve the condition of patients with HF. These guidelines are regularly updated and recommend differential diagnosing and treatment strategies with dissemination programs for physicians. The perception of the physician toward the diagnosis and management of HFpEF may affect optimal care. Narrowing the existing gap between knowledge and practice might support new initiatives in the optimization of diagnosing, treatment, and management strategies in patients with HFpEF.