Mycobacterium tuberculosis complex, remains a serious global public health problem, representing one of the main causes of death from infectious diseases. About one quarter of the world’s population is infected with Mtb and has a latent TB infection (LTBI). According to the World Health Organization (WHO), an LTBI is characterized by a lasting immune response to Mtb antigens without any TB symptoms. Current LTBI diagnoses and treatments are based on this simplified definition, although an LTBI involves a broad range of conditions, including when Mtb remains in the body in a persistent form and the immune response cannot be detected. The study of LTBIs has progressed in recent years; however, many biological and medical aspects of an LTBI are still under discussion. This review focuses on an LTBI as a broad spectrum of states, both of the human body, and of Mtb cells. The problems of phenotypic insusceptibility, diagnoses, chemoprophylaxis, and the necessity of treatment are discussed. We emphasize the complexity of an LTBI diagnosis and its treatment due to its ambiguous nature. We consider alternative ways of differentiating an LTBI from active TB, as well as predicting TB reactivation based on using mycobacterial “latency antigens” for interferon gamma release assay (IGRA) tests and the transcriptomic analysis of human blood cells.Tuberculosis (TB), caused by bacilli from the  Mtb antigens in the absence of any clinical manifestation of the disease -protein acyltransferase) genes involved in lipid metabolism. The deletion of MTS0997 leads to a slower growth of Mtb and M. bovis in a lipid deficient medium. The overexpression of MTS0997 and MTS1338 leads to a slowdown in cell growth in the culture and is significantly higher in dormant “nonculturable” Mtb cells [Mtb are as follows: mcr7 regulates virulent properties, 6C inhibits growth, MTS0997 regulates lipid metabolism, and MTS1338 controls the expression of genes in the DosR regulon. MTS0997 and MTS1338 are present only in the M. tuberculosis complex; they regulate the stress response during the phagocytosis of the pathogen in the host [The transition to dormancy is not a random process for  process . The dorconstant . The levironment . MTS0997tb cells . To summthe host .Mtb cells either by immunity (both innate and acquired) or after anti-TB chemotherapy, where viable Mtb cells do not present, although immunological signs of infection may be observed; LTBI—caused by viable Mtb cells, which have immunological signs of TB infection with no clinical, radiological, or microbiological evidence of TB, and will not progress to TB in the case of strong immunity; incipient—likely to progress to TB in the absence of control, but is not yet manifested either clinically, radiologically, or microbiologically; subclinical form—there are no have clinical symptoms of TB, but it already causes radiological or microbiological changes; and active TB—which causes clinical symptoms with radiological changes/microbiological signs compatible with TB. These stages can interchange with different dynamics. The increase in the disease severity usually correlates with an increase in symptoms, a low likelihood of spontaneous self-healing, a weakened or poor immune response, and an increased bacterial excretion. However, spontaneous recovery is possible at any of the stages described above [The binary model of TB infection with a latent infection and an active disease has been criticized . Insteaded above .Mtb infection (as the presence of persistent Mtb cells in the body) and an LTBI. The definition of an LTBI, as a diagnosed immune response to Mtb antigens in the absence of any TB signs, is generally accepted [Neither authors of previously published papers in the LTBI area, nor WHO recommendations, differentiate between the accepted ,68,69. Aaccepted ,70. The accepted ,72.(1)If they have both a positive (IGRA) test and a positive tuberculin skin test (TST) and are in contact with TB patients;(2)Mtb, and a contact was vaccinated with bacille Calmette-Guerin (BCG)) or when the TST size is larger than 15 mm ;If they have a negative IGRA, but their TST size is larger than 5 mm, and they are in close contact with TB patients (when the source of infection excretes (3)If they have a negative IGRA and their TST size is smaller than 10 mm, but their TST size increases and becomes larger than 10 mm (the difference is not less than 6 mm);(4)BCG vaccination, or TST is greater than or equal to 15 mm in case of BCG vaccination).If they have a negative IGRA and positive TST  accelerate the reactivation of TB infection significantly [Most studies show that some high-risk factors  blockers, hemodialysis, and close contact with ficantly ,79,80. Tficantly ,81.BCG, as well as non-tuberculous mycobacterium (NTM) infections in patients with HIV, lead to false positive results [Immunological methods occupy a special place in the diagnosis of an LTBI . Previou results ,85. Fals results ,87.Mtb [BCG and NTM [Modern tests are based on the evaluation of the levels of IFN-γ production after stimulation with the recombinant ESAT-6/CFP-10 fusion protein, with the early secreted virulence factors encoded in the RDI (region of difference) of Mtb . These t and NTM . The spe and NTM ,89. More and NTM . Reactio and NTM ,91. It h and NTM , leading and NTM ,92.Mtb metabolism and life cycle, either in vitro, in animal models, or in humans, made it possible to find Mtb antigens that could be efficient in the discrimination of an LTBI , hypoxia , vitamin C exposure , and intra-phagosomal infection in naive and activated macrophages  [These antigens were identified as potent latency-related biomarkers for an LTBI diagnosis. Some of them were identified after modeling in vitro hypoxia , nutritited mice . The Mtb Rv2873) .Mtb Rpf antigens may be suitable for the screening of T-cell responses to TST-positive people. In this case, IFN-γ production was observed in response to Rv1009 (rpfB), Rv1884c (rpfC), Rv2450c (rpfE), and, to a smaller degree, Rv0867c (rpfA) [Mtb-specific ESAT-6/CFP-10, Rv0867c, and Rv2624c had accurately identified 73% of TB patients and 80% of the non-TB cases after cross-validation [Moreover, it was shown that c (rpfA) . Among tc (rpfA) . In anotlidation .Rv1733c, a putative membrane protein, was shown to be preferentially recognized by T-cells from people with an LTBI, compared to TB patients . AntigenDosR-controlled antigen—alpha-crystallin (Rv2031)—was shown to enhance the efficiency of an LTBI diagnosis in a TB endemic setting via a strong increase in IFN-γ, TNF-α, and interleukin-10 levels. The increased levels of these cytokines were suggested as biomarkers of an LTBI [DosR regulon, were purified as recombinant proteins and were tested for differentiating people with an LTBI from people with TB in Europe, Africa, South America, and India. In all cohorts, these antigens mostly stimulated a response in people with an LTBI, compared to patients with TB (either active or treated) [The  an LTBI . The anttreated) . The Rv2treated) . A two-htreated) .Mtb survival under hypoxia and nonreplicating conditions [Rv0847, produced under the control of the copper inducible five-locus regulon, is specific to virulent mycobacterial species. Its role was shown to be the ability to sense the host macrophage environment, and it might be crucial to nditions . The antnditions .p = 0.125), 67.9% to 87.5% (p < 0.001), and 85.7% to 92.9% (p = 0.125), respectively [Using Rv2659 alone did not detect any difference between TST-negative people and TST-positive healthy people. However, a significant difference was shown between TST-positive people and TB patients. Rv2660 did not reveal a statistical difference between TST-negative controls and TST-positive healthy people, whereas the difference between the TST-negative controls and TB patients was significant . Rv1985cectively .Mtb showed their potent efficiency in identifying people with an LTBI and distinguishing them from healthy controls or active TB patients based on the IFN-γ production levels. Moreover, in some cases, they were efficient even in differentiating between an LTBI and a recently acquired Mtb infection and, thus, could be considered as novel candidates to expand the IGRA test antigen panel.The so-called “latency antigens” of Mtb cells (actively dividing and dormant) and the immune cells [Mtb, preventing the reactivation of TB [The TB process is a dynamic relationship between ne cells . In an Lon of TB ,108. Curon of TB ,109. Thion of TB ,110. If on of TB ,110.Chemoprophylaxis in adults should focus on high-risk groups . The effectiveness of isoniazid monotherapy has been shown for the first two groups . At the To increase the effectiveness and reduce the duration of chemoprophylaxis, various schemes are used in world practices. The effectiveness of therapy with isoniazid and rifapentine once a week for 12 weeks has been shown for homeless people . A studyMtb persisters, and helps to shorten the anti-TB therapy duration significantly [An equally important criterion in the LTBI treatment is the total cost of chemotherapy. One study in Australia analyzed the cost of two LTBI treatment regimens: 9 months of isoniazid monotherapy and 12 weeks of isoniazid/rifapentine regimens. The cost of one completed course of treatment was AUD 601 for the first regimen– and AUD 511 for the second . Thus, fficantly . The comficantly ,126,127.Mtb cells: alanine dehydrogenase, isocitrate lyase, cysteine synthase CysM, adenosine-5′-phosphosulfate reductase, DevS and DosT oxygen sensors involved in dormancy response, lysine ε-aminotransferase, enoyl-acyl carrier protein reductase (InhA), decaprenyl-phosphoryl-ribose 2′-epimerase (DprE1), mycocyclosin synthase (Cyp121), and extracellular zinc metalloprotease 1 (Zmp1). Details of the above-mentioned compounds targeting nonreplicating Mtb cells, a description of their chemical structures, and the possible modes of action are provided in the review by Campaniço et al. “Addressing latent TB: new advances in mimicking the disease, discovering key targets, and designing hit compounds” [However, new anti-TB compounds are under exploration. They are targeted at blocking the enzymes that remain active during the dormant state of mpounds” .Mtb cells, thus promoting their resuscitation. The Mtb genome contains five genes encoding sequences for the Rpf A-E proteins [Mtb resuscitation and TB reactivation, they are of great interest as potential targets for the development of anti-TB drugs aimed at treating LTBIs and preventing TB reactivation [Mtb bacilli [Another possible direction of the anti-TB drug design is preventing TB reactivation via inhibiting resuscitation promoting factors (Rpfs), which are hydrolases with muralytic activity that are able to cleave the cell wall of dormant proteins . Since ttivation . These p bacilli .An essential component of the WHO End TB Strategy is TB prevention by identifying and treating people with an LTBI based on positive immunological tests. In countries with a high TB burden, the LTBI treatment is the most important strategy for TB control. Nevertheless, a point of view, that the attention to LTBIs is exaggerated, also exists . About 1Mtb antigens. However, the immune response to Mtb antigens will take place even after all live Mtb bacilli have been eliminated either by immunity or due to their treatment with anti-TB drugs [Mtb for one year reduced the incidence of TB by 60–70% over the next 9 years, the TST of these people remained positive [Although immunological tests are commonly used to identify individuals with an LTBI, they only reflect the presence of immune responses to TB drugs . If the positive . Anotherpositive . Similarpositive .Mtb cells in the body, or by its stimulation with mycobacterial antigens, while viable bacilli have long been eliminated. The results of testing with QFT-TB, before starting chemoprophylaxis in people with a suspected LTBI in a country with low TB endemicity, was positive in 30.8% (148/481) of the total number of subjects, in 66.9% (111/166) of people who moved from TB-endemic countries, in 71.4% (20/28) of those who were previously treated for TB, and in 100% (15/15) of those diagnosed with active TB. These people were given chemoprophylaxis, and then the QFT-TB test was repeated. In 35/40 (87.5%) and 22/26 (84.6%) of people, the QFT-TB test was positive after 3 and 15 months of treatment, respectively [Moreover, the use of IGRA tests to diagnose the effectiveness of treatments does not shed light on the cause of the immune response. It remains uncertain as to whether it is caused by the persistence of ectively . In anotectively . In one ectively .These results are consistent with the development of a strong immunological memory, which is enhanced by prolonged exposure to the antigen , but it However, despite the facts above, computer modelling has shown that if at least 8% of people with an LTBI received treatment annually, the total global incidence of TB would be fourteen times lower by 2050 compared to 2013, even in the absence of additional measures .Transcriptomic analyses make it possible to determine the individual gene expression signatures of the host in response to a pathogen. It has already been shown that the levels of gene expressions change in diseases of various etiologies, including psoriasis , parasitCXCL10, ATP10A, and TLR6, as well as the DOCK9, EPHA4, and NPC2 genes from peripheral blood samples, incubated with PPD, significantly differed in patients with active TB, people with an LTBI, and healthy individuals from England, Africa, and Brazil [NPC2 in one of the patients who did not receive chemoprophylaxis subsequently led to the development of active TB, but after treatment, it decreased significantly [CXCL10, DUSP3, FCGR1A, GBP5, SEPT4, ANKRD22, BATF, FCGR1B, FCGR1C, GAS6, GBP1, GBP6, LHFPL2, S100A8, SCARF1, and SERPING1, which were the most strongly associated with TB [SERPING1, TRMT2A, GBP2, SDR39U1, FCGR1B, and TUBGCP6) whose expression levels demonstrated the highest predictive power for the risk of TB reactivation.This method has already shown to be both high in specificity and sensitivity for differentiating between active TB and LTBIs in adults. It was observed that changes in the expression levels of the d Brazil ,142. Morficantly . In 2018 with TB . Then, i with TB . It contCXCL10, DUSP3, FCGR1A, GBP5, SEPT4, ANKRD22, BATF, FCGR1B, FCGR1C, GAS6, GBP1, GBP6, LHFPL2, S100A8, SCARF1, and SERPING1 genes were most frequently involved in immune responses against pathogens:The products of the CXCL10 encodes a proinflammatory chemokine of the Cysteine X Cysteine (CXC) family, which is involved in a wide range of processes, including leukocyte chemotaxis, the differentiation and activation of peripheral immune cells, and the regulation of cell growth, apoptosis, and the modulation of angiostatic effects;DUSP3 encodes a dual specificity phosphatase 3 (which dephosphorylates phosphoserine and phosphotyrosine residues), which negatively regulates mitogen-activated protein kinases;FCGR1A (or CD64), FCGR1B, and FCGR1C (pseudogene) encode high-affinity receptors for fragments crystallizable (Fc) of immunoglobulin-γ, as well as playing a role in the activation of innate and adaptive immunity, and they are present in monocytes and macrophages;GBP1, GBP5, and GBP6 encode guanylate-binding proteins involved in the activation of the inflammasome and autophagolysosome assemblies in response to a bacterial infection;SEPT4 encodes septin, a guanosine triphosphate hydrolase (GTPase) that is a part of filaments, and plays a role in the recognition of pathogen cells;ANKRD22 encodes a protein with an unidentified function containing an ankyrin repeat domain;BATF encodes a transcription factor of the Activator protein 1 (AP-1) family, which controls the differentiation of immune cells;GAS6 encodes a specific growth arresting protein, and is a ligand of Axl receptor tyrosine kinase, tyrosine-protein kinase receptor TYRO3, and tyrosine-protein kinase MER, as well as inhibiting innate cellular immunity;LHFPL2 encodes a protein of the tetraspan transmembrane family responsible for cell adhesion, motility, activation, and proliferation;S100A8 encodes a protein of the calprotectin family that regulates immune responses and inflammation, and this gene also activates the nicotinamide-adenine dinucleotide phosphate (NADP) oxidase complex, inducing its assembly;SCARF1 encodes a scavenger receptor family protein that mediates the binding and degradation of acetylated low density lipoproteins;SERPING1 encodes an inhibitor of plasma C1 proteases responsible for the inhibition of the complement system.Some researchers believe that the correct transcriptomic analysis should include the study and comparison between the gene expression levels of the individual immune cell populations: mononuclear cells , neutropMtb are the main reservoir of TB; therefore, their identification and treatment is an important strategy. It can effectively reduce the risk of TB and the number of potential sources of infection in the future [BCG vaccine does not provide the adequate prevention of pulmonary TB in adolescents and adults, allowing the infection to spread [Mtb cell and immune cell physiology, as well as to shed light on their states during an LTBI and the reactivation of the infection.The goals of the WHO TB strategy are to reduce TB mortality by up to 95%, and morbidity by up to 90%, by 2035. People latently infected with e future ,112. Thee future  more into spread . It is,


GNG13, CD163, C1QA, TLR2, SST, C1QB, ITGB2, CCR5, CRH, and TAC1), four central regulatory transcription factors , and four microRNAs . Hub gene–drug interaction analysis found four drugs  that could be potential drugs for VaD treatment. Together, our results may contribute to understanding the underlying mechanisms in VaD and provide potential targets and drugs for therapeutic intervention.Vascular dementia (VaD) is considered to be the second most common form of dementia after Alzheimer’s disease, and no specific drugs have been approved for VaD treatment. We aimed to identify shared transcriptomic signatures between the frontal cortex and temporal cortex in VaD by bioinformatics analyses. Gene ontology and pathway enrichment analyses, protein–protein interaction (PPI) and hub gene identification, hub gene–transcription factor interaction, hub gene–microRNA interaction, and hub gene–drug interaction analyses were performed. We identified 159 overlapping differentially expressed genes (DEGs) between the frontal cortex and temporal cortex that were enriched mainly in inflammation and innate immunity, synapse pruning, regeneration, positive regulation of angiogenesis, response to nutrient levels, and positive regulation of the digestive system process. We identified 10 hub genes in the PPI network ( Vascular dementia (VaD) is a neurocognitive disorder that also has other characteristics such as behavioral symptoms, locomotor abnormalities, and autonomic dysfunction . VariousBioinformatics analysis of transcriptomic data is used widely to identify biomarkers, therapeutic targets, and the mechanisms of various diseases, including dementia . Key modA schematic diagram of the workflow of the bioinformatics analyses is shown in 1 | > 1. Volcano plots were used to visualize the DEGs in frontal cortex or temporal cortex versus the matched controls. A Venn diagram was used to identify the overlapping DEGs between the two brain regions (We used the Gene Expression Omnibus (GEO) dataset, GSE122063, which contains gene expression profiles of the frontal cortex and temporal cortex samples from patients with AD, VaD, and matched controls . In the 1 . To iden regions .2  enrichment analysis under the three main GO categories, biological process, molecular function, and cellular component, and pathway enrichment analysis of the overlapping DEGs between the two brain regions were performed on the web-based portal Metascape2 . The Kyo2 , Reactom2 , and Wik2  were use2 . P-value3 to construct a PPI network; a combined score of >0.7 was set as the cut-off criterion of statistical significance  database, version 11.0ificance . Cytosca network . Ten gen5  and several drug databases   ChEMBL) . The 10 The differential expression analysis detected 128 upregulated and 183 downregulated genes in the frontal cortex , and 131The protein class over-representation analysis of proteins encoded by the overlapping DEGs classified them into different groups according to their functions and activities . The resGNG13, CD163, C1QA, TLR2, SST, C1QB, ITGB2, CCR5, CRH, and TAC1 . Heterotrimeric G proteins, which consist of alpha, beta, and gamma subunits, function as signal transducers for the seven-transmembrane, G protein-coupled receptors. GNG13, which encodes a gamma subunit, is expressed in taste, retinal, and neuronal tissues, and the GNG13 protein is involved in the gustatory signal transduction pathway by interacting with proteins containing PDZ domains  family, whose members are significant pattern recognition receptors of the innate immune system, initiating inflammatory cascades by recognizing pathogen- and damaged-associated molecular patterns. TLR2 was found to play a pivotal role in inflammation after ischemic brain injury  mouse model by inhibiting activation of the PKA-CREB signaling pathway  and four miRNAs  as regulators of the hub genes in VaD. FOXC1 is involved in vascular development processes, including arterial specification and angiogenesis regulation, and may play a role in small vessel disease, which is the primary pathology underlying vascular cognitive impairment . Cyclic  pathway . GATA2, ed in AD , but itsThe reduced level of miR-27a-3p found in the cerebrospinal fluid of patients with AD indicated it as a candidate biomarker for AD . A recenClinicalTrials.gov registry (see footnote 8). Although none of the 22 drugs have been used directly to treat VaD, four of them  could be potential drugs for VaD treatment. Maraviroc, cenicriviroc, and PF-04634817 are all CCR5 antagonists and efalizumab is an IGTB2 antagonist. Maraviroc and cenicriviroc were reported to improve cognition in HIV-infected individuals with cognitive impairment through their antiretroviral and anti-inflammatory effects  can be found in the article/JS, WW, and LZ conceived and designed the study, analyzed the data, wrote the manuscript, and contributed to the designs of the methods used in this study. All authors have read and agreed to the publication of this version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YZ has declared a shared parent affiliation with the authors at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Coptidis rhizome dry extracts (CR) can be combined (HHCR) as a herbal product. Previous studies have demonstrated that HHCR has antitussive and expectorant effects in animal models of respiratory disease. However, the therapeutic effects of HHCR on respiratory diseases in humans have not been well-studied. Therefore, we aimed to clarify the effectiveness of HHCR in patients with chronic bronchitis and bronchiectasis. Methods: This was a multicenter , open-label, prospective, single-arm, observational study. Consecutive patients with chronic bronchitis and bronchiectasis were included. Patients were orally treated with HHCR daily for 12 weeks. St. George’s Respiratory Questionnaire (SGRQ) scores and bronchitis severity scores (BSS) were measured at baseline and at the end of the 12-week study. Results: In total, 376 patients were enrolled, of which 304 were finally included in the study, including 236 males and 68 females with a median age of 69 years (range: 37–88 years). After 12 weeks of HHCR treatment, there was a significant improvement in SGRQ score  and a significant reduction in BSS . During the study, 14 patients concomitantly used an inhaled corticosteroid and 83 patients used an inhaled bronchodilator. HHCR also had significant positive effects on these patients in terms of SGRQ score and BSS. No serious adverse drug reactions occurred during HHCR treatment. Conclusions: treatment with HHCR improved the SGRQ score and BSS in patients with chronic bronchitis and bronchiectasis. HHCR may be a new therapeutic option for chronic bronchitis and bronchiectasis. Large-scale, randomized, double-blind, placebo-controlled clinical trials are warranted.Background: Hederacoside C from ivy leaf dry extracts (HH) and berberine from  Bronchiectasis is a chronic respiratory disorder associated with poor quality of life (QOL) and frequent exacerbations. There are some international guidelines on treatment of bronchiectasis, including The European Respiratory Society (ERS) and British Thoracic Society (BTS) guidelines. Chronic bronchitis may degrade lung function and increase the rate of exacerbation, resulting in clinical consequences such as aggravate health-related QOL and increase the possibility of mortality . The sevCoptidis rhizome (CR) are consumed in Asian countries, including China, Japan, Malaysia, Singapore, and India, but very little is used in European countries [MUC5AC gene, which is primarily involved in mucin production in chronic inflammatory airway diseases, and some studies have shown that berberine exhibits bronchodilatory and antimicrobial activities [Large quantities of ountries . The theountries . Kim et ountries . reportetivities . Hederactivities .HHCR is a herbal product consisting of HH from ivy leaf dry extracts and berberine from CR dry extracts. HHCR is approved in the Republic of Korea for the relief of cough and sputum formation due to acute respiratory infection and inflammation. The research on CR and HH has mostly focused on molecular mechanisms using animal studies ,6,8,9. AThis 12-week, open-label, multicenter, prospective, single-arm, observational study was conducted at 10 university teaching hospitals in the Republic of Korea. The first patient was enrolled on 25 September 2017, and the last patient was examined on 28 June 2018. The study protocol was designed by the investigator steering committee and presented to all study locations thereafter. All investigators were trained before the trial to ensure reliable data quality, with special emphasis on understanding the protocol. The study was conducted in accordance with the Declaration of Helsinki, and Good Clinical Practice Guidelines. The protocol was approved by the institutional review board at each study center, and all patients gave written informed consent before participating in any study procedures.®; Ahn-Gook Pharmaceutical Co., LTD., Seoul, Korea) for 12 weeks. At every visit, adherence to the study regimen was assessed. The study flowchart is shown in Patients were seen at the initial visit, at week 4 after enrollment, and at week 12 of the study; any unscheduled visits were recorded. Patients were treated orally with fixed dose 15 mg of HHCR three times daily  that started prior to the study, including the use of short-acting bronchodilators, long-acting bronchodilators such as long-acting β2-agonists and long-acting muscarinic antagonists, inhaled corticosteroids (ICS), and methylxanthines such as theophylline and doxofylline, was permitted to continue during the study period, whereas the use of mucoactive drugs, antitussives, antibiotics, and systemic corticosteroids was prohibited to avoid any potential confounding effects on clinical endpoints.Patients aged ≥35 years, regardless of whether they were smokers or non-smokers, were eligible if they had been diagnosed with chronic bronchitis based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines  or broncMycobacterium tuberculosis , pneumonia, history of active peptic ulcer disease or intestinal malabsorption, congestive heart failure categorized as class 2 or higher according to the New York Heart Association, severe neurological disease, severely impaired hepatic or renal function, immunocompromised status, suspected or known hypersensitivity to the study product or any of its excipients, rare hereditary problem of fructose intolerance, pregnancy or active lactation status, poor reliability , and poor compliance.Exclusion criteria were: acute exacerbation of chronic bronchitis, continuous treatment with systemic corticosteroids, active infection due to The primary endpoint was the change in St. George’s Respiratory Questionnaire (SGRQ) score between the baseline and week 12. The SGRQ is disease-specific instrument designed to assess of quality of life. It is consists of 50 items divided into three components: symptom component, activity component, and impact component. Each score of components and total score are calculated. The SGRQ is displayed with a score ranging from 0 to 100, where 0 indicates the best quality of life related to health, and the higher score means a poorer quality of life.The secondary endpoint was the change in Bronchitis Severity Score (BSS) between the baseline and week 12. The BSS was developed to measure an appropriate outcome of bronchitis . The scaPatients were first seen at a screening visit during which inclusion/exclusion criteria, medical and medication history, and the status of exacerbations were checked, and the results of a physical examination, chest radiograph, electrocardiography (ECG), laboratory tests, and pregnancy test as well as the SGRQ score and BSS were recorded. Patients who met the inclusion criteria were asked to sign the informed consent form. After the start of the study, patients returned to the investigation center at weeks 4 and 12. At each visit, the following information was recorded: physical examination results, pregnancy test results, concomitant medications , occurrence of spontaneous adverse events (AEs), and level of compliance. At week 12, the SGRQ and BSS were assessed again.Safety assessments included the recording of AEs, laboratory tests, blood pressure measurements, and 12-lead ECG. All adverse events or concurrent illnesses during the study were documented in the clinical record form. Major adverse cardiovascular events  were evaluated and classified by an independent, blinded adjudication committee. AEs were evaluated and presented descriptively. An AE was defined as any untoward medical occurrence in a patient who was administered the study drug, regardless of whether it was considered to be related to the study drug. An AE was considered serious if it was either immediately life threatening or resulted in persistent or significant disability, prolonged hospitalization, or death.t-test. All statistical analyses were performed using SAS for Windows software . A value of p < 0.05 was considered to denote significance.All data are presented as medians or the means ± standard deviations (SDs) for continuous variables and as numbers with percentages in parentheses for categorical variables. The primary positive outcome in the study would be an improvement in the SGRQ score and BSS after 12 weeks of HHCR treatment. Changes from baseline to the endpoints were analyzed using the paired Of 377 eligible patients with chronic bronchitis or bronchiectasis who were recruited, one patient was excluded during the screening period due to withdrawn consent. Of the 72 (19.2%) patients who discontinued treatment, 27 (7.2%) were lost to follow-up, 15 (4%) had protocol violations, 14 (3.7%) withdrew consent, 5 (1.3%) had AEs, 5 (1.3%) were non-compliant, and 6 (1.6%) discontinued for other reasons.Patient demographics and baseline characteristics are summarized in p < 0.0001) after 12 weeks of treatment (p <0.0001) and impact domains  after 12 weeks. The activity domain score also improved , but the difference was not statistically significant  and the mean was 32.52 ± 16.93 (SD) at baseline. At week 12, a median score of 25.64  and a mean score of 29.08 ± 15.16 were reported. Patients treated with HHCR exhibited a statistically significant and clinically relevant improvement from baseline in terms of SGRQ total score, with median reduction in score of −2.37 and a mean reduction of −3.44 ± 10.97 (reatment . Of the nificant .p < 0.0001) after 12 weeks of treatment (p < 0.0001), sputum production , auscultation , chest pain while coughing , and dyspnea  (p < 0.0001). A scatter plot of the variation in BSSs vs. variation in SGRQ scores is shown in The total median and mean BSS at baseline were 7.0  and 7.16 ± 2.63, respectively, and the total median and mean scores at week 12 were 4.50  and 4.72 ± 2.45, respectively. Patients treated with HHCR exhibited a statistically significant and clinically relevant improvement from baseline in terms of total BSS, with a median score reduction of −2.0 and a mean score reduction of −2.45 ± 2.36 (reatment . Of the  0.0001) . The corp = 0.0115) after 12 weeks of treatment. Statistically significant and clinically relevant improvements after 12 weeks were seen in the symptoms  and impact  domain scores. The activity domain score improved , but the improvement was not statistically significant  and the mean score was 37.66 ± 18.54 at baseline. At week 12, a median score of 29.86  and a mean score of 34.51 ± 17.74 were reported. Patients treated with HHCR exhibited a statistically significant and clinically relevant improvement from baseline in terms of SGRQ total score, with a median score reduction of −1.71 and a mean score reduction of −3.15 ± 13.31 (nificant .p < 0.0001). Of the BSS components, statistically significant and clinically relevant improvements were seen after 12 weeks in terms of scores related to coughing , sputum production , auscultation , chest pain while coughing , and dyspnea   and 6.88 ± 2.75, respectively, and the median and mean total scores at week 12 were 4.0  and 4.55 ± 2.35, respectively. The change in total BSS from baseline to week 12 reflected a statistically significant and clinically relevant improvement, with a median score reduction of −2.0 and a mean score reduction of −2.33 ± 2.66 ( 0.0001) .Based on pooled analysis of safety data, 30 AEs were reported by 28 patients (9.21%) from the treatment population during the 12-week study. Most AEs were mild in severity and generally well-tolerated. The most frequent AEs were respiratory symptoms, including coughing, dyspnea, rhinorrhea, and sore throat, followed by gastrointestinal symptoms such as nausea, gastric irritation, and heartburn. Neurologic symptoms such as insomnia and myalgia were also reported. Of the 30 AEs, only four were determined by the investigators to be related to the study drug. A summary of reported AEs is shown in In all, three patients (0.99%) reported three serious AEs, including two cases of dyspnea and one increase in prostate specific antigen. However, two of the serious AEs were determined by the investigators to be unrelated to the study drug. Only one patient (0.3%) discontinued HHCR treatment because of a serious AE (dyspnea). All the AEs improved spontaneously during treatment with HHRC; therefore, these AEs were not causally related to HHCR treatment. During the 12-week observation period, no MACE or fatal outcomes were reported. No abnormal findings were observed in chest radiographs, ECG outputs, or laboratory tests, including tests for full blood count, blood chemistry, and urinalysis during the study period.Bronchiectasis is a chronic respiratory disease, radiologically characterized by abnormal and permanent expansion of the bronchi, and clinical syndromes such as sputum production, cough, and bronchial infection . Sputum Chronic bronchitis result from goblet cells producing and secreting excessive mucus. By luminal obstruction of small airways, epithelial remodeling, and alteration of airway surface tension, it contributes to aggravating airflow obstruction and finally make the airway vulnerable to collapse. Despite the clinical sequelae, there is only limited information on the pathophysiology of chronic bronchitis and goblet cell hyperproliferation of COPD, and there are insufficient alternatives for treatment . ImpairmThere has been a persistent effort to use traditional natural products as respiratory medications to alleviate coughing and sputum production for thousands of years. HHCR is a natural medicine reported to suppress pulmonary inflammation, and it has been shown to have expectorant and antitussive effects in animals . QOL impThe results of our study indicate that HHCR is able to reduce the respiratory symptoms of patients with chronic bronchitis and bronchiectasis. Treatment with HHCR improved both SGRQ score and BSS. Moreover, subgroup analysis of the changes in SGRQ score and BSS among patients concomitantly treated with a bronchodilator showed that the SGRQ score and BSS improved in this patient group compared with the group with no concomitant bronchodilator treatment. Previous study has suggested that HHCR is a potential useful therapeutic option for respiratory diseases . HoweverHH is a triterpene saponin and an active ingredient derived from ivy leaf extracts. It exhibits biological properties such as expectorant and bronchospasmolytic effects, and hence has been therapeutically applied for the treatment of chronic inflammatory bronchial conditions and productive coughs ,23,24. CChronic bronchitis and bronchiectasis with acute exacerbations are difficult to treat, resulting in high morbidity and mortality. Unfortunately, no foundational treatment agent has been identified. Some mucolytics and expectorants such as N-acetylcysteine, erdostein, and ambroxol are used for reducing mucus secretion and promoting mucus clearance, but there have been concerns about their effectiveness, limitations, and side effects . Our stuNevertheless, the study has some limitations. First, this was an open-label, single-arm, observational trial, and treatment with a bronchodilator and ICS was permitted. This open design might have biased the results, and the use of concomitant treatment is a possible confounding factor. However, there were no significant differences in the use of bronchodilator treatments from baseline to week 12 in all enrolled patients, and HHCR treatment was uniformly administrated to all patients in the study. Additionally, regarding the bronchodilator, out study’s bronchodilator uses were not specialized as short-acting or long-acting. Therefore, the difference in effect between each type of bronchodilator is limited. Despite this limitation, this pilot clinical trial is the first one performed in patients. Further large-scale placebo-controlled studies are warranted. Second, objective parameters of airway inflammation such as cytokines, chemokines, and sputum inflammatory cells were not estimated. However, the focus of this study was to determine an individual’s health-related QOL change using validated and widely accepted instruments such as the SGRQ and BSS. Third, our study’s design investigated 12 weeks only, so further further safety evaluations for extended periods and patient outcomes needed . However, clinical improvements and no serious AEs after 12 weeks of treatment could be considered reasonable treatment goals for chronic bronchitis and bronchiectasis. Finally, we did not evaluate correlations between HHCR and lung function/clinical parameters. Further large-scale clinical trials are needed to elucidate these correlations.Despite some limitations, the results of this observational, real-world trial revealed that patients with chronic bronchitis and bronchiectasis experienced considerable and clinically relevant improvements in health-related QOL, with improvements in the total BSS score and all five domain scores as well as the SGRQ total score and all three domain scores after treatment with HHCR. The changes in SGRQ score and BSS among patients concomitantly treated with a bronchodilator showed that the SGRQ score and BSS improved in this patient group compared with the group with no concomitant bronchodilator treatment. The potential attenuation of symptom progression associated with chronic bronchitis and bronchiectasis reported in this study suggests that HHCR may be a reasonable therapeutic agent for further prospective placebo-controlled trials.


Due to the high prevalence of malnutrition among hospitalized patients, screening and assessment of nutritional status should be routinely performed upon hospital admission. The main objective of this observational study was to evaluate the prevalence of and the risk for malnutrition, as identified by using three nutritional screening tests, and to observe whether some anthropometric and functional parameters used for nutritional evaluation were related to these test scores.This single-center observational study included 207 patients admitted from the emergency department for hospitalization in either the internal medicine or surgery units of our institution from September 2017 to December 2018. The prevalence of malnutrition among this patient sample was evaluated by using the Nutritional Risk Screening (NRS-2002), the Subjective Global Assessment (SGA) and the Global Leadership Initiative on Malnutrition (GLIM) criteria. Body mass index (BMI), bioimpedance analysis (BIA), handgrip strength (HGS) and calf circumference (CC) assessments were also performed.According to the NRS-2002, 93% of the patients were at no risk or at low nutritional risk (NRS score < 3), and 7% were at a high nutritional risk (NRS score ≥ 3). On the other hand, according to the SGA, 46.3% of the patients were well-nourished (SGA-a), 49.8% were moderately malnourished (SGA-b), and 3.9% were severely malnourished (SGA-c). Finally, according to the GLIM criteria, 18% patients were malnourished. Body weight, body mass index (BMI), phase angle (PhA), CC and HGS were significantly lower in the patients with NRS scores ≥ 3, SGA-c and in patients with stage 1 and stage 2 malnutrition, according to the GLIM criteria.The NRS-2002, the SGA and the GLIM criteria appear to be valuable tools for the screening and assessment of nutritional status. In particular, the lowest NRS-2002, SGA and GLIM scores were associated with the lowest PhA and CC. Nevertheless, a weekly re-evaluation of patients with better screening and assessment scores is recommended to facilitate early detection of changes in nutritional status. Disease-related malnutrition among hospitalized patients is a major public health issue in both industrialized and developing countries around the world, with a reported prevalence between 20 and and 50%, according to differences in study populations, assessment methods, and hospital settings , 2. MalnTo identify patients who are at risk for malnutrition or who are malnourished upon hospital admission, nutritional screening should be performed as a part of standard care. Screening tests must be quick and easy to apply; in already malnourished patients, a thorough nutritional assessment should also be performed , 3.The European Society for Clinical Nutrition and Metabolism (ESPEN) recommends using the Nutritional Risk Screening 2002 (NRS 2002) to screen adults upon hospital admission .The Subjective Global Assessment (SGA) is a nutrition assessment test used worldwide that allows for the grading of nutritional status in different conditions; it includes a more complex set of questions and must be conducted by specially trained professionals.Objective methods for nutritional assessment include anthropometry  body composition evaluated by BIA and functional tests.In literature, several studies have already evaluated the relation between parameters predicting malnutrition and poor clinical outcomes . The pre2 suggest chronic malnutrition and are associated with poor outcomes and high mortality rates  seem to be associated with malnutrition or high nutritional risk , 12.Bioelectrical impedance analysis (BIA) is a non-invasive, low-cost and simple method widely used to assess body composition; BIA-derived phase angle (PhA) is a reliable indicator commonly used for nutritional assessments , 14. It Finally, handgrip strength (HGS) is a validated and easily implemented measure of muscle strength that has been frequently used for clinical purposes, particularly in recent years. Low HGS values are associated with long hospitalization durations and high re-admission rates , 18.In order to standardize the diagnosis of malnutrition, the Global Leadership Initiative on Malnutrition (GLIM) criteria, were also applied. The GLIM criteria are now considered as the best ones for diagnosing malnutrition in adults in clinical care settings and are based on a two-step model for the risk screening and diagnostic assessment of malnutrition .Anyway, different screening tools may lead to different results for the risk of malnutrition, due to their different clinical significance . No reseOur study aims to evaluate the prevalence of and the risk for malnutrition, as determined by the NRS-2002 screening test, the SGA and by GLIM criteria, in patients upon hospital admission from an emergency department and in hospitalized patients in the internal medicine unit and the surgery unit of a single center and, to observe whether some specific parameters used for nutritional assessment were related to NRS-2002 and SGA test scores.The sample size was calculated considering the primary endpoint and assuming, according to the literature , 7, an eAll Caucasian adult patients coming from the emergency unit and admitted to either the internal medicine unit or the surgery unit were recruited in our study from September 2017 to December 2018.All patients were consecutively screened for inclusion.The exclusion criteria were transfer from an intensive or critical care unit, pregnancy or breastfeeding, and inability to communicate.Fifty patients refused to participate, 80 presented exclusion criteria, and 30 were excluded for other reasons; finally, 207 patients were eligible for inclusion in the study.Informed consent was obtained just after admission, and the tests were performed during the first 48 h after admission by the same staff members to reduce the risk of bias during the measurement process. The study staff members included three dietitians and a biologist with expertise in clinical nutrition; they were all trained, according to the good clinical practice guidelines, with theoretical and practical sessions, to perform the screening tests and the measurements.All data were collected and stored following SQL Server Security Best Practices.The NRS-2002 takes into account weight loss, BMI, food intake reductions and impaired general conditions. The disease severity score considers current clinical conditions as well as chronic diseases with acute complications . Scores from 0 to 3 correspond to these conditions. The total score is obtained from the nutritional and disease severity evaluation and adjusted for age in patients older than 70 years (+ 1 point), according to Kondrup's recommendations , 21. An The SGA includes the patient's history , a brief physical examination  and the clinician's overall evaluation of the patient's status. Each patient is classified as well-nourished (SGA-a), suspected or moderately malnourished (SGA-b), or severely malnourished (SGA-c) .The GLIM criteria are composed of three phenotypic and two etiologic criteria. A combination of at least one phenotypic , and one etiologic (reduced food intake/ assimilation and disease burden) criterion is necessary to make diagnosis of malnutrition.2 or age ≥70 and BMI at admission <22 kg/m2; (b) age <70 and admission BMI <18.5 kg/m2 or age ≥70 and admission BMI <20 kg/m2  moderate malnutrition or (b) severe malnutrition were also employed: (1) % weight loss: (a) > 5% or (b) > 10% and; (2) BMI: (a) if age <70 and BMI at admission <20 kg/m20 kg/m2 .2) (2) presence of inflammation acute disease/injury related, another GLIM phenotypic criterion, because all patients coming from an Emergency Department.Body composition measurements to assess muscle mass deficit, for the etiologic GLIM criterion the following criteria were used based on data that were available: (1) fat free mass index , specific formulas that involved knee height were used .2 and was divided into the following categories:BMI was calculated as weight/height2 indicated underweight; 18.5 ≤ BMI > 25 kg/m2 indicated normal weight; and BMI ≥25 kg/m2 indicated overweight—obesity  was measured by using a dynamometer .Bedridden subjects moved their arms parallel to the trunk, grasped the dynamometer, and applied the maximum force possible with each hand.The measurement was repeated three times, in 1-min intervals to avoid fatigue, with each hand or on one side only in patients undergoing intravenous therapy and in those with other limitations; the mean and the maximum of the three recorded measurements were recorded in kilograms (kg).The cut-off points used to diagnose dynapenia were < 27 kg for men and < 16 for women .Bioimpedance analysis (BIA) was performed using a Human Im Touch device  that measures resistance, impedance and PhA at 50 kHz. With the subject lying supine, four surface electrodes were placed on the non-dominant wrist and ankle. The patients were tested in their rooms after at least 15 min of lying during the first 48 h post hospital admission.2)/resistance ohm) using the Sun BIA equation  and fat mass (FM), were assessed by the BI-Index .t test for unpaired data was used to evaluate the differences among groups with different test scores, and the chi-squared test was used to evaluate the relative frequencies within different test score groups. A normality test for variables has been performed, where applicable, and statistical analysis done accordingly. The Man-Whitney test was used for non-parametric variables.Student's p values are based on two-sided tests and compared data among different groups to a significance level of 5%.All reported Independent associations among variables were assessed with stepwise regression analysis.Statistical analysis was performed using SPSS for Windows, version 27.0 .n = 125 were older than 60 years), with a BMI range of 26.2 ± 5.4 kg/m2 . Fifty-five percent (n = 114) of the patients was hospitalized for a cardiovascular or hepatic impairment, 23% (n = 48) for a diagnosis of oncologic disease and 22% (n = 46) for a gastrointestinal disease.Our study included 207 Caucasian patients, 110 (53%) males and 97 (47%) females, aged between 18 and 85 years  patients were at no risk or at low nutritional risk, and 14 (7%) were at high nutritional risk. On the other hand, the SGA assessed those 96 (46.3%) patients were well-nourished, 103 (49.8%) were moderately malnourished, and 8 (3.9%) were severely malnourished.According to the GLIM criteria, 37 (18%) patients were malnourished; in particular 18 patients had moderate malnutrition (stage 1) and 19 patients severe malnutrition (stage 2) .The patients with NRS scores ≥3  were older and had lower body weight, and BMI than those with NRS scores < 3 ; moreover, they had lower CC, HGS and PhA than those patients with NRS scores <3 .n = 2) had a BMI < 18.5 kg/m2, 64.3% (n =9) had a CC <31 cm, 79% (n = 11) had a PhA < 5°, and 79% (n =11) had a HGS < 16 for female and <27 for males.Specifically, for patients with NRS-2002 scores ≥ 3, 14%  were older and had lower body weights than those in the SGA-b  and the SGA-a  groups. No significant differences were observed in height or BMI .Both patients classified as moderate and severe malnourished according to the GLIM criteria, had significantly lower PhA and CC than not malnourished patients. Also, after adjusting the analysis by age, the differences in the results were confirmed.n = 2) in the SGA-c group had a BMI <18.5 kg/m2, 50% (n = 4)of them had a CC < 31 cm, 87.5% (n = 7) had a PhA < 5°, and 88% , a high prevalence of patients with low CC, PhA and HG values was observed.p = 0.016); while, regarding to NRS-2002 ≥ 3, in the multiple regression analysis, the presence of a low calf circumference (p = 0.015) was the major independent predictor.About the SGA-c score, the liner regression model highlights that only PhA is an independent prognostic factor , while according to the SGA, 49.8% of patients were moderately (SGA-b) and 3.9% (SGA-c) severely malnourished. Based on the GLIM criteria, when using all combinations of the two-criteria diagnosis identified about 18% of patients to be malnourished. When severe malnutrition was considered, GLIM identified a higher proportion (around 51%) than SGA (around 4%). Based on prevalence alone and compared to SGA, GLIM seems to represent a larger proportion of overall malnutrition but is more likely to identify a person as severely malnourished.This apparent inconsistency in results may be primarily because the SGA classified patients into one of three levels: , whereas the NRS 2002 addresses two categories only . Although both the NRS 2002 and the SGA consider the metabolic stress of disease and changes in food intake, the NRS-2002 classifies metabolic stress using numerical scores, while the SGA depends on the investigator's experience to indicate the metabolic stress of disease .Moreover, the NRS-2002 contains questions that indicate recent or acute changes in nutritional status (percent of weight loss in the last three months) and age, while the SGA includes questions related to the detection of chronic malnutrition . This variety of questions could also be responsible for the different results , 30–32.The NRS-2002 is a fast, easy and useful screening tool that seems well-suited to be applied in an acute phase to patients coming from an emergency department; however, the exclusive use of the NRS-2002 might underestimate the real incidence of malnutrition in hospitals .Soon after patient stabilization, the SGA could be integrated into nutritional evaluations. In September 2018, in order to build a global consensus on the diagnostic criteria for malnutrition, the GLIM criteria were proposed. According to these criteria, patients were categorized into two groups: moderate (stage 1) and severe (stage 2) malnutrition. This study showed that both patients with moderate and severe malnutrition had lower PhA and CC compared with those without malnutrition.At our best knowledge, the GLIM criteria had been still not validated in patients coming from an Emergency Department.PhA is the most clinically established impedance parameter and has been suggested to be an indicator of cellular health, and nutritional status, and be highly predictive of impaired clinical outcome and mortality in a variety of disease.PhA represent a clinically feasible approach to body composition, free from equation inherent errors and necessary assumption. It been shown to be a superior indicator of survival and outcome and is generally used as screening tool for identification of patients at nutritional risk. BIVA provides more detailed information on hydration and cell mass integrity and should be considered as an assessment and monitoring tool .Low PhA values predict poor outcomes, long hospital stays and morbidities , 33. In HGS seems to detect muscle loss, fiber quality, and functionality earlier while providing a better evaluation of nutrition repletion after therapy . It may In our study, very low HGS values (<16 in F and <27 in M) were found in patients with SGA-c scores and in those with an NRS-2002 ≥ 3; this association has also been confirmed by several studies showing lower HGS among patients at high nutritional risk, as evaluated by both screening tests , 37.In summary, in our study, low SGA (SGA-c) scores were clearly associated with the lowest values of PhA, while CC (p.0.015) has the major prognostic role in patients with NRS 2002 ≥3, and as for the GLIM criteria the same happens with the lowest PhA values (p. 000) and CC.Low PhA and HGS values were observed in all Caucasian hospitalized patients evaluated, independent of their SGA and NRS-2002 and GLIM scores. These results confirm the need for close monitoring of all patients during hospitalization  to detect possible changes in clinical and nutritional status early on.Study limitations are the relatively heterogeneous population, the small sample size, the data results from a single hospital, the lack of detailed clinical information on the evaluated patients and of their clinical outcomes; these last two deficiencies are linked to the nature of the observational study. Another study limitation is that we have not used BIVA to better define if low PhA was linked to malnutrition, fluid overload or both. Anyway, this study had not the primary aim to obtain detailed information on hydration.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethical Committee of the Federico II University Hospital. The patients/participants provided their written informed consent to participate in this study.FP, GD, and FC conceived and designed the study. ES, OD, CD, and MN collected research data. OD, DM, and MM analyzed the results. ES and MM drafted the manuscript. LS and FP made critical revisions to the manuscript for important intellectual content. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mus mattheyi shows completely differentiated sex chromosomes, representing an uncommon evolutionary situation among mouse species. We have performed a detailed analysis of the location of proteins involved in synaptonemal complex assembly (SYCP3), recombination  and sex chromosome inactivation (γH2AX) in this species. We found that neither synapsis nor chiasmata are found between sex chromosomes and their pairing is notably delayed compared to autosomes. Interestingly, the Y chromosome only incorporates RPA and RAD51 in a reduced fraction of spermatocytes, indicating a particular DNA repair dynamic on this chromosome. The analysis of segregation revealed that sex chromosomes are associated until metaphase-I just by a chromatin contact. Unexpectedly, both sex chromosomes remain labelled with γH2AX during first meiotic division. This chromatin contact is probably enough to maintain sex chromosome association up to anaphase-I and, therefore, could be relevant to ensure their reductional segregation. The results presented suggest that the regulation of both DNA repair and epigenetic modifications in the sex chromosomes can have a great impact on the divergence of sex chromosomes and their proper transmission, widening our understanding on the relationship between meiosis and the evolution of sex chromosomes in mammals.X and Y chromosomes in mammals are different in size and gene content due to an evolutionary process of differentiation and degeneration of the Y chromosome. Nevertheless, these chromosomes usually share a small region of homology, the pseudoautosomal region (PAR), which allows them to perform a partial synapsis and undergo reciprocal recombination during meiosis, which ensures their segregation. However, in some mammalian species the PAR has been lost, which challenges the pairing and segregation of sex chromosomes in meiosis. The African pygmy mouse  Meiosis is a specialized type of cell division essential for the transmission of chromosomes across generations . During Mus musculus, it was reported that these DSBs are introduced by the SPO11α isoform, acting only in the PAR at the end of zygotene [While most chromosomes follow this general pattern, sex chromosomes in mammalian male meiosis are prone to present exceptions to some of these processes. In most species the X and Y chromosomes are highly differentiated in size and gene content ,9. Neverzygotene ,22. Accozygotene ,21. The zygotene , and frozygotene ,25. Finazygotene ,27,28.Although the presence of the PAR is widespread in mammals, some species present completely differentiated sex chromosomes  ,34,35,36Mus mattheyi. The genus Mus is composed of a large number of species that are very similar phenotypically, but show great genetic divergence [Nannomys [n = 16 to 2n = 36 [M. mattheyi [Nannomys, with an ancestral-like 2n = 36 karyotype where all chromosomes are acrocentric, meaning no centric fusions between autosomes nor between sex chromosomes and autosomes [Mus minutoides, which differs to other pygmy mice because in this species a sex-autosome fusion between both the X and the Y and the autosomal pair 1 have restored a large neo-PAR that synapses and recombines at meiosis [In order to gain insights into the meiotic behavior of achiasmate sex chromosomes, here we have studied male meiosis in the African pygmy mouse vergence . AfricanNannomys ,49, whic 2n = 36 . M. mattmattheyi  is one outosomes . It was utosomes ,55,56,57M. mattheyi were bred in captivity at the CECEMA facilities of Montpellier University. The colony was established and maintained under standard conditions as previously reported [reported . Males w2HPO4, 1.7 mM KH2PO4, pH 7.4) overnight at room temperature in a humid chamber. The following primary antibodies and dilutions used were: rabbit anti SYCP1 , 1:100; rabbit anti SYCP3 (Abcam 15093), 1:100; mouse anti SYCP3 (Abcam 97672), 1:100; mouse anti γH2AX , 1:1000; rabbit anti histone H2AX phosphorylated at serine 139 (γH2AX) (Abcam 2893), 1:1000; rabbit anti RPA2 (Abcam 10359), 1:100; rabbit anti RAD51 , 1:50; mouse anti-MLH1 , 1:100; and a human serum that recognizes centromere proteins , 1:100. After rinsing in PBS, the slides were incubated with secondary antibodies diluted to 1:100 in PBS for one hour at room temperature: donkey anti-mouse, donkey anti rabbit and goat anti-human, conjugated with Alexa 350, Alexa 488, Alexa 549 , Cy3 or Dylight 649 . Slides were counterstained with DAPI, when needed, and mounted with Vectashield .We prepared spermatocyte spreads and squashes following the procedures previously described ,60. SlidZ axis. Images were captured with an Olympus DP61 camera and processed with Adobe Photoshop CS software. The length of the SC was measured using ImageJ software. For the squashes, several optical sections were recorded for each cell. The stack files were processed with ImageJ to make three-dimensional reconstructions as previously described [Observations were made on an Olympus BX61 microscope equipped with a motorized plate in the escribed ,45.M. mattheyi have a meiotic karyotype of 2n = 36, with 17 autosomal bivalents plus the X and Y sex chromosomes, all of them being acrocentric (n = 11 spermatocytes), while the Y chromosome is small and represents 4.35% (±0.70) of the total SC.Males of ocentric . The X iThe progression of prophase-I was characterized through the immunolocalization of SYCP3, a component of the axial-lateral elements (AEs/LEs) of the SC, and γH2AX (histone H2AX phosphorylated at serine 139), a histone modification that marks chromosomal regions that present DNA damage and/or have not completed synapsis and is also a typical marker of MSCI. The localization and morphological pattern of these two proteins allow the precise discrimination of the different stages in mammalian meiosis . In leptM. mattheyi is quite similar to that described in M. musculus meiosis [M. mattheyi is rather different. The AEs of the X and Y chromosomes become distinguishable from the rest of chromosomes by late zygotene, and they appear as thinner filaments not involved in synapsis (n = 51) at this stage, sex chromosomes lie apart from each other with no contact of either AEs or γH2AX signal  in late zygotene, 56.0% (n = 75) in early pachytene and 43.0% (n = 200) in mid pachytene. When present, the number of RPA foci on the Y chromosome is limited, ranging from 1 to 5 . This indicates that the dynamics of RPA in this chromosome is clearly different from the autosomes and the X chromosome.The distribution and dynamics of RPA in d humans ,64. A lan = 131), 34.26% of early pachytene (n = 108) and 10.62% of mid pachytene spermatocytes (n = 339) have RAD51 foci on the Y chromosome. When present, a single focus of RAD51 is observed. Altogether, these results indicate that most spermatocytes do not exhibit RAD51 on the Y chromosome. Moreover, after double localization with RPA, we found that RAD51 foci were only located in the Y chromosome when RPA foci were also present, although, as observed in autosomes, both kinds of foci rarely colocalize  has been extensively studied for decades [M. mattheyi presented here offers new clues to understand how the alterations of sex chromosome pairing, synapsis and recombination, which cause severe meiosis impairment in the house mouse, can be overcome in closely related species, allowing sex chromosomes to evolve to complete differentiation.The meiotic behavior of sex chromosomes in the laboratory and wild house mouse . However, this may not be a terminal situation for sex chromosomes in mice within the Nannomys subgenus. This should be placed in the context of the high karyotypic variability described in this group [Nannomys are regularly engaged in new translocation events with autosomes, as described in M. minutoides. In this species the completely differentiated X and Y chromosomes have been translocated to chromosome 1, generating a neo-XY chromosome pair with a large neo-PAR, which now combines the coexistence of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate association in the differentiated segments [Asynaptic sex chromosomes are rare in mammals, but are common in some groups of rodents such as gerbils and voles ,45,46,78romosome ,37. Thisromosome ,82,83. Tromosome . Subsequromosome . This woromosome ,45,46,85is group . Indeed,segments ,86. Intesegments ,88,89. Tsegments . NeverthM. musculus laboratory strains causes meiosis impairment, severe fertility defects, sex chromosome mis-segregation and aneuploidy [Loss of synapsis between sex chromosomes challenges two critical aspects of their meiotic behavior: DNA repair and pairing. This is especially intriguing, taking into account that hampering either pairing or recombination between sex chromosomes in euploidy ,90. TherM. musculus the induction of DSBs in the PAR is extremely regulated, relaying on the action of the specific SPO11α isoform [As indicated above, in most mammals, sex chromosome synapsis is dependent on programmed DNA damage that is subsequently repaired through homologous recombination. In  isoform  and the  isoform ,24,25. F isoform . Soon af isoform . Accordi isoform .M. mattheyi we did not observe the structural modifications encompassed by the PAR in M. musculus, even under super-resolution fluorescence microscopy [M. mattheyi would be regulated by the same mechanisms acting in M. musculus, but at least two differences are relevant about the subsequent repair of DNA on the Y chromosome. First, induction of damage is not followed by the expected accumulation of DNA repair proteins. Up to 46% and 70% of spermatocytes do not incorporate RPA or RAD51 on the Y chromosome, respectively. The absence of these proteins suggests that a strikingly high proportion of spermatocytes may repair DNA in the Y chromosome using a pathway different from homologous recombination. Second, the noticeable difference in the Y chromosome between the proportion of RPA foci compared to that of RAD51 suggests that upon DNA resection, this damage would not be repaired by the canonical homologous recombination pathway using the sister chromatid as template, as previously suggested [Caenorhabditis elegans have suggested that single strand-annealing can indeed be involved in the repair of the single X chromosome in male meiosis [In croscopy . Howeveruggested ,26,27,28 meiosis .M. mattheyi, slight changes in the regulation of DNA repair, or the diversion of this repair to non-recombinogenic pathways, could have a similar effect. In both cases, the homologous recognition of X and the Y chromosomes would be hampered and thus recombination would be abrogated. Under this scenario, it is possible to speculate that loss-of-function mutations in genes like SPO11α or AKRD31, slight changes in the microsatellites that drive DSB production in the PAR [These striking features of DNA repair on the Y chromosome open interesting possibilities to understand the cessation of recombination between the X and Y chromosomes. Although the deletion of the PAR is the most plausible cause to explain the asynaptic and achiasmate nature of sex chromosomes in  the PAR , or modiM. mattheyi is delayed. How do these homology-independent pairing mechanisms work? Sex chromosomes could just move on the nuclear envelope as a consequence of the general movements of chromosomes during pachytene [M. musculus models [Albeit sex chromosomes do not synapse, pairing between them becomes necessary for ensuring proper segregation. As observed in other species with asynaptic sex chromosomes ,31,37,38achytene ,94 and ts models ,24,25. TM. mattheyi sex chromosomes could display a similar mechanism. However, we have not found any signs of SYCP3 involvement in this association. Although the distal end of the Y chromosome seems to be oriented to an interstitial region of the X chromosome, we have not observed a physical contact between these SYCP3 labelled regions. Instead, the sex chromosomes seem to remain associated just through a chromatin contact. The association through heterochromatin regions between asynaptic sex chromosomes has already been suggested for the species Psammomys obesus [M. mattheyi does not exhibit such heterochromatin regions.Different mechanisms have been suggested for ensuring the segregation of achiasmate sex chromosomes. In marsupials, the persistence of the dense plate up to metaphase-I was proposed to act for that purpose . The dens obesus ,33 and fs obesus ,36. HoweM. mattheyi seems to represent just a remnant of the sex body formation during pachytene, which entails the condensation of chromatin and the incorporation of a number of epigenetic modifications [M. musculus models with defects in either synapsis or recombination of sex chromosomes [M. mattheyi is the persistence of γH2AX in the sex chromosomes through the first meiotic division. This histone modification has been observed to persist in the achiasmate sex chromosomes of other mammalian species. This is the case of the Mongolian gerbil Meriones unguiculatus and the Mediterranean pine vole Microtus duodecimcostatus [M. minutoides after their translocation to an autosome [M. minutoides chromosomal fissions seem to have restored the original (unfused) condition of sex chromosomes [Therefore, the association of sex chromosomes through chromatin interactions in ications ,76,95,96omosomes ,24,25. Iomosomes ,97. Howecostatus ,46. The autosome . This reomosomes . The preM. mattheyi with M. musculus offers the possibility to undertake genomic comparisons that could reveal slight but significant differences with consequences for the outcome of meiosis. It is particularly interesting that small changes in the regulation of DNA repair or epigenetic modifications of sex chromosomes can have a profound impact on the subsequent transmission and evolution of these chromosomes, particularly the Y chromosome. Thus, the final stages of sex chromosome differentiation could be achieved more easily than anticipated, provided that accurate mechanisms for pairing and segregation are working. Obviously, meiosis emerges as an important factor to be considered in the study of sex chromosome evolution.The results presented here offer new clues about the behavior of sex chromosomes during meiosis. The close evolutionary proximity of


Primary care physicians have been present on the frontline during the ongoing pandemic, adding new tasks to already high workloads. Our aim was to evaluate burnout in primary care physicians during the COVID-19 pandemic, as well as associated contributing factors.Cross-sectional study with an online questionnaire disseminated through social media, applying the snowball technique. The target population was primary care physicians working in Portugal during the first outbreak of the COVID-19 pandemic. In addition to sociodemographic data, the questionnaire collected responses to the Copenhagen Burnout Inventory (CBI), the Resilience Scale and the Depression, Anxiety, and Stress Scales (DASS-21). Data were collected from May 9 to June 8, 2020, a period comprising the declaration of a national calamity and then state of emergency, and the subsequent ease of lockdown measures. Levels of burnout in 3 different dimensions , resilience, stress, depression, and anxiety were assessed. Logistic regression analyses were conducted to identify factors associated with burnout levels.Among the 214 physician respondents, burnout levels were high in the 3 dimensions. A strong association was found between gender, years of professional experience, depression and anxiety, and burnout levels.Physician burnout in primary care is high and has increased during the pandemic. More studies are needed in the long term to provide a comprehensive assessment of COVID-19’simpact on burnout levels and how to best approach and mitigate it during such unprecedented times. For Portuguese physicians, that day marked the beginning of a great challenge that would soon be declared a global pandemic by the World Health Organization (WHO).2 It is considered a relevant occupational health hazard among healthcare workers (HCW) and has a significant impact on professionals, patients and health institutions. Burnout was already a problem in Portugal prior to COVID-19, with 43.6% of Portuguese physicians reporting high levels of burnout.3Burnout is defined in the 11th Revision of the International Classification of Diseases (ICD-11) as a syndrome resulting from chronic workplace stress that has not been successfully managed. It is characterized by 3 dimensions: feelings of energy depletion or exhaustion; increased mental distance from one’s job, or feelings of negativism or cynicism related to one’s job; and reduced professional efficacy.4 the pandemic has brought new stressors possibly contributing to physician burnout, such as fears of becoming infected or infecting a relative, lack of appropriate personal protective equipment, lack of access to up-to-date information and communication, limited time with family and friends, reductions in economic revenue, and increased demands from childcare and household tasks.57As the COVID-19 pandemic increases pressure on healthcare systems worldwide, physician burnout levels are also expected to rise. With a total of 32 500 confirmed cases and 1410 deaths from COVID-19 as of May 31,810Around the world, general practitioners and family doctors were on the frontline fighting the spread of infection. In the Portuguese health care system, the family doctor acts as a gatekeeper; while maintaining this role during the pandemic, they had to adjust and add new activities to their daily schedules  while keeping up-to-date with new evidence and guidelines on the novel coronavirus, all in addition to their normal preventive and curative medical activity.11 In a recent study of 2008 HCWs in Portugal during the pandemic, where burnout was measured by the validated Portuguese version of the Copenhagen Burnout Inventory (CBI),12 high burnout levels were reported by 52.5% of participants for personal burnout, 53.1% for work-related burnout, and 35.4% for patient-related burnout.13 Another work focusing on the levels of burnout in HCW in Italy during the COVID-19 pandemic used the Maslach Burnout Inventory (MBI), whose subscales assess levels of emotional exhaustion, depersonalization and personal accomplishment. This study revealed high levels of emotional exhaustion in 41% and high levels of depersonalization in 27%.14 Wu et al. specifically compared levels of burnout in frontline vs. other HCWs in Wuhan, China to find that the first had significantly lower levels of burnout and were less worried about becoming ill compared to those in the “usual ward” group,15 highlighting that all HCWs should be considered when designing well-being policies, irrespective of their position.In a study to understand the impact of COVID-19 around the world that included a total of 2707 participants from 60 countries, 51% of HCWs reported burnout.16Kristensen et al. developed the CBI based in the concept that burnout is not just fatigue or exhaustion, it is the attribution of these feelings to specific domains of one’s life, as a result CBI is a tool with 3 subscales: personal, work, and client-related burnout. The personal burnout subscale measures feelings of physical, emotional, and mental fatigue and exhaustion. The work-related burnout subscale assesses the symptoms that respondents attribute to work. The client-related burnout subscale describes the aforementioned feelings that respondents attribute to their work with clients.To our knowledge, no study to date has focused on assessing primary care physicians’ burnout levels in addition to stress, depression, anxiety, and resilience during the COVID-19 outbreak, nor the associated contributing factors.Cross-sectional study with an online questionnaire disseminated through social networks, spread using the snowball technique.The study population was Portuguese-speaking primary care physicians working in Portugal during the COVID-19 pandemic.® Forms, was spread as a web link through social networks and institutional mailing lists. All participants gave their informed consent to participate in this study and anonymity was assured. The first section of the questionnaire addressed sociodemographic data. Psychological variables collected in the following sections included the Copenhagen Burnout Inventory (CBI), the Resilience Scale and the Depression, Anxiety, and Stress Scale (DASS-21).Data were collected from May 9 to June 8, 2020, a period comprising the declaration of a national calamity, the subsequent state of emergency, and the ease of lockdown measures that followed. The questionnaire, developed using Google12 This is a 19-item scale with 3 subscales: personal (6 items), work (7 items), and patient-related burnout (6 items). Each item is answered on a 5-point Likert scale . The score for each subscale is the average of item scores within the subscale, ranging from 0 to 100. A score of 50 or higher in any of the subscales was considered high-level burnout.16 These subscales demonstrated high internal consistency .16 In the current study, α was .91, .89, and .89 for personal burnout, work-related burnout, and patient-related burnout, respectively.The validated Portuguese version of the CBI was used to measure burnout.17 The total score corresponds to the sum of the 25 items, thus ranging from 25 to 175. A score below 121 is considered “low resilience,” 121 to 145 points “moderate resilience” and higher than 145 “high resilience.” The validated Portuguese version presented high internal consistency, with α = .8918 and α = .94 in a medical sample19 and α = .95 in our study.The Resilience Scale is composed of 25 items, each with a 7-point Likert response scale ranging from 1 (strongly disagree) to 7 (strongly agree).21 The depression subscale is scored as follows: normal (0-9), mild (10-13), moderate (14-20), severe (21-27), and extremely severe (28 or more). In the anxiety subscale, scores are as follows: normal (0-7), mild (8-9), moderate (10-14), severe (15-19), and extremely severe (20 or more). In the stress subscale, scores are as follows: normal (0-14), mild (15-18), moderate (19-25), severe (26-33), and extremely severe (34 or more). For DASS-21, the values of α in our study were .90, .84, and .90 for the depression, anxiety, and stress subscales, respectively. For the present analysis, each subscale was categorized into normal and not normal .DASS-21 is composed of 3 subscales evaluating depression, anxiety, and stress, each with seven 4-point Likert scales .® 2016 (USA) spreadsheet and statistical analyses were performed using SPSS® Statistics .Data from the survey were exported to a Microsoft ExcelQ1; Q3] . Normal distribution was assessed with visual inspection of the histograms.Categorical variables were described using absolute and relative frequencies, n (%). Quantitative variables with a normal distribution were described with mean , where a value higher than .7 was considered acceptable.P ≤ .20 in the simple logistic regression were included in the multiple logistic regression analyses. Only the significant variables were maintained in the final multivariate models for each burnout dimension. To convey the results of logistic regressions odds ratios (OR), 95% confidence intervals [95% CI], and P-values are presented. The final models were evaluated using the Hosmer & Lemeshow test of adequate fit. P ≤ .05 was considered significant.Separate multiple logistic regressions were performed for personal, work-related, and patient-related burnout. The independent variables to include in each multiple logistic regression were chosen by conducting simple logistic regressions with each variable. All variables correlating with the outcomes with Considering there are 8000 Portuguese physicians in a Primary Healthcare specialty  we planned a sample size between 192 and 367 respondents, considering the most conservative scenario (a proportion of 50%), for a level of confidence of 95% and an error margin between 5% and 7%.225 primary care physicians answered the questionnaires, but 11 were excluded from the analysis for being either retired or absent from work due to disease or leave. Therefore, a total of 214 respondents were considered . Of these 214 respondents, 95.3% were working on the frontline  during the pandemic. The sociodemographic characteristics of the participants are described in High levels of burnout were found for the 3 dimensions: 65.9% for personal burnout, 68.7% for work-related burnout, and 54.7% for patient-related burnout . ResilieParticipants reported levels of depression (67.3%), anxiety (69.6%), and stress (61.7%) which fell within the normal range on the measurement tool .P = .019). Having worked for 6 to15 years was also significantly associated with higher odds of patient-related burnout  compared to those with 5 or fewer years of practice. A reduction in monthly income was significantly and inversely correlated with patient-related burnout.Results for the multiple logistic regression analyses for personal, work-related, and patient-related burnout are presented in Tables 3P = .001), work-related burnout , and patient-related burnout , compared with normal levels.Higher levels of depression were significantly associated with higher levels of all 3 burnout dimensions: personal burnout  and work-related burnout .Higher levels of anxiety were also significantly associated with higher levels of personal burnout .28Another study assessing burnout during the pandemic found that younger age and being female were independent determinants of burnout, similar to our results.The use of the CBI scale to assess burnout is one of the strengths of our study: in addition to its excellent psychometric properties and validation for the Portuguese population, we found this scale to be particularly appropriate for the population being studied, since the CBI is composed of 3 different domains that also reflect primary care physicians’ daily activities.29Having used a convenience sample, our results may not be generalized to other populations and contexts. Additionally, we cannot rule out that the physicians who decided to answer our survey might be different from those who did not respond, nor can a self-reporting bias be excluded. Our sample consisted of 80.8% female physicians, which could suggest a gender-biased response, which may reflect the female predominance in family medicine in Portugal: in 2019, 62.2% of family doctors were female, with 73.2% females in the 36 to 40 years age group.The questionnaire may not address other important contributors to burnout. Notably, it is not possible to establish a causal relationship between the pandemic and our findings.30 Our findings reinforce that strategies to counteract physician burnout during a pandemic need to be further investigated. Our workgroup suggests next steps should include, at an organizational level, involving physicians in designing guidelines and contingency plans and also in implementing physician’s access to feedback channels. To feel better prepared, they should receive rapid basic training and have the opportunity to talk to experts. A supportive network should be created, including childcare, transportation, and lodging.7 Emotion management strategies and self-care should also be endorsed, encompassing rest, work breaks, sleep, shift work, fatigue, and healthy lifestyle behaviors.31 Physicians with depressive and anxious symptoms are at increased risk of personal and work-related burnout; therefore, specific interventions should be aimed at identifying and helping this group. Specific programs to prevent burnout should also be implemented for physicians just starting their careers, such as coping and self-care strategies for medical residents. More studies are needed in the long term to provide a comprehensive assessment of the impact of the COVID-19 pandemic and to further evaluate it as a contributing cause for burnout, for instance, in a study using a sequential mix-method approach.Burnout has negative impacts on physicians, patients, and healthcare organizations.


Bardet-Biedl syndrome (BBS) is an autosomal recessive pleiotropic ciliopathy, which includes multi-organ clinical manifestations. The known genes involved in the development of the disease account for the causality in about 80% of the examined cases.in silico tools for their pathogenicity. Patients’ phenotypes were also assessed. We investigated two Iranian unrelated clinically diagnosed BBS patients, using a targeted next-generation sequencing panel consisting of 18 known BBS genes. The detected variants were investigated in the pedigree and studied using BBS9 gene in each patient, c.2014C>T, p.Gln672Ter and c.673_674insAA, p.Gln225GlnfsX10. The variants were segregated in the corresponding pedigree and were authenticated to obtain enough evidence to be categorized as pathogenic variants.  Novel homozygous variants were detected in  Patients with truncating mutations in the same gene seem to show similar phenotypic features. Detection of novel and family-specific mutations is typically expected in the genetic hereditary diseases in Iran, which can finally lead to prevent the recurrence of the disease in the consanguineous marriages Bardet-Biedl syndrome (BBS) is one of the most pleiotropic ciliopathies, which is mostly considered to be inherited in an autosomal recessive pattern; however, there is evidence that BBS presents with an oligogenic nature , 2. BBS The incidence of BBS ranges from nearly 1 in 125,000 in North America and Europe to 1 in 17,000 in the Bedouins of Kuwait, and 1 in 3700 in the Faroe Islands. The higher incidence is observed in geographically isolated or highly consanguineous populations -12. BBS1, BBS10, BBS2, BBS9, MKKS, BBS12, MKS1, BBS4, BBS7, TTC8, ARL6, BBIP1, BBS5, CEP290, IFT27, LZTFL1, SDCCAG8, TRIM32, and WDPCP (in order of attribution) can cause BBS, which can account for the causality in about 80% of the examined cases . Next-generation sequencing was performed on an Illumina HiSeq 2500 NGS platform  provided by Beijing Genomics Institute .The DNA isolation was performed from the whole blood using the GeneAll® Exgene™ Blood SV DNA purification kit according to the manufacturer’s instructions. Target region capturing was performed with a custom-designed Nimblegen chip for coding regions and the exon-intron boundaries of 18 BBS-related genes  tools  to determine the nucleotide and amino acid conservation status and effect of the sequence variant at the nucleotide and amino acid level, including determination of the effect of the variant on the primary and alternative gene transcripts and other genomic elements, as well as the potential impact of the variant on the protein , 1000 Genomes Project (1000G), and dbSNP], disease databases , and sequence databases  for any previously reported record. The variants were also analyzed by several computational , or M13F-pUC(-40) (tagged to reverse primer). Exon 7 was sequenced by a forward PCR primer.The variant identified by NGS analysis in each patient was confirmed by Sanger sequencing . Segregation analysis of the detected variants was performed in each pedigree by polymerase chain reaction (PCR) followed by Sanger sequencing. The clinical characteristics of the identified variants are summarized in BBS9 . Characteristics of the identified variants and The results of Sanger sequencing validation of the detected variants in the patients and the sequencing results of the same point in their parents are demonstrated in BBS9 variants, c.2014C>T, p.Gln672Ter in patient one and c.673_674insAA, p.Gln225GlnfsX10 in patient two, were confirmed with Sanger sequencing and were segregated in the pedigree in an autosomal recessive pattern. These variants have not been previously reported for their pathogenicity. Both alterations have been expected to result in a truncated protein product and activate the nonsense-mediated decay (NMD) mechanism. NMD acts as a surveillance system and protects the cell against the toxic effect of a protein with a premature termination codon (PTC). To do so, the PTC should be located in the NMD target region  assembly  guidelines, both identified variants are classified as "pathogenic" variants. The clinical diagnosis of the disease was confirmed and the health care provider could use the molecular testing information in clinical decision-making. Furthermore, variants classified as “pathogenic” according to ACMG guidelines, can be used in pre-implantation genetic testing (PGD) and prenatal diagnosis (PND) in the future pregnancies of the pedigree. BBS1, BBS10, BBS2, BBS9, MKKS, BBS12, MKS1, BBS4, BBS7, TTC8, ARL6, BBIP1, BBS5, CEP290, IFT27, LZTFL1, SDCCAG8, TRIM32, and WDPCP (in order of attribution) have been identified to cause BBS, which account for the causality in about 80% of the examined cases  contribution for BBS9 (BBS9 (Several reviews have estimated a ~ 6% contribution of patients . Interesfor BBS9 . HoweverS9 (BBS9 . TherefoGenerally, studies with a large sample size have not been performed on Iranian BBS patients yet. Because of a high rate of consanguineous marriages in the Iranian population, novel and family-specific mutations are typically expected to be detected in the case studies of the genetic hereditary diseases in Iran, similar to the current study. In Conclusion, our study reported two novel variations in BBS9, a nonsense variant in exon 19 and a frame-shift variant in exon 7, in two unrelated Iranian families with an affected member in each. The variants were categorized as “pathogenic”. The clinical diagnosis was confirmed and the families were referred for comprehensive genetic counseling before PGD and PND in their future pregnancies. These authors contributed equally to this work and are considered as co-first-authors.The authors report no conflict of interest