In plants, salicylic acid (SA) is a hormone that mediates a plant’s defense against pathogens. SA also takes an active role in a plant’s response to various abiotic stresses, including chilling, drought, salinity, and heavy metals. In addition, in recent years, numerous studies have confirmed the important role of SA in plant morphogenesis. In this review, we summarize data on changes in root morphology following SA treatments under both normal and stress conditions. Finally, we provide evidence for the role of SA in maintaining the balance between stress responses and morphogenesis in plant development, and also for the presence of SA crosstalk with other plant hormones during this process. Recognized as the sixth plant hormone in 1992 , SA has It is worthy of note that, in roots, SA content and its dynamic during development may differ from that in shoots ,6,7,8, waim1 [Naphthalene hydroxylase G (NahG) gene, encoding salicylate hydroxylase that inactivates SA by converting it to catechol [Lotus japonicus plants expressing NahG, demonstrate enhanced root growth [The phenotypic analysis of SA deficient/accumulating lines and SA-treated plants provides insight into the role of SA in plant growth and development. However, the data on changes in root morphology in SA mutants are scarce and often contradictory. For example, decreased root length is reported in SA-accumulating Arabidopsis mutants ,10,11 anaim1  and plancatechol . In contt growth . These ct growth ,15.In contrast to SA mutants, changes in root morphology after an SA treatment are described in numerous papers. In our review, we analyzed more than 100 studies on SA treatments in 40 plant species, with SA doses ranging from 10 fM to 10 mM, to survey the impact of SA on root system biomass and architecture under normal conditions and in response to stressors. In this paper, we discuss various aspects of SA’s regulation of root growth and development, including changes in root morphology after SA treatments, the molecular basis of these changes, and the impact of SA in root responses to abiotic stress and other environmental changes.SA metabolism has been comprehensively described in numerous reviews, for example, ,18,19,20A wide number of regulators control SA biosynthesis . Among tO-β-d-glucose (SAG) and salicylic acid glucose ester (SGE). Uridine diphosphate (UDP)-glycosyltransferases UGT74F1 and UGT74F2 perform the conversion to the former, while only UGT74F2 is involved in the catalysis to the latter. The carboxyl group can be also methylated by the S-adenosyl-L-methionine (SAM)-dependent methyltransferase, BA/SA carboxyl methyltransferase 1 (BSMT1), producing methyl salicylate (MeSA), the form of SA that has increased membrane permeability. SA hydroxylation by SA-5 and SA-3 hydroxylases generates 2,3-DHBA and 2,5-DHBA dihydroxybenzoic acids. Gretchen Hagen 3.5/WESO 1 (GH3.5/WES1) and another unknown GH3 family enzyme convert SA into salicyloyl-L-aspartate (SA-Asp). Some of these conjugated forms of SA may also be glycosylated. Inactive SA forms can be stored until they are required to increase the active pool of free SA; alternatively, some of them may be subjected to SA catabolism. SA is often spread via apoplast . TheThe NONEXPRESSOR OF PATHOGENESIS-RELATED GENES (NPR) are the SA receptors . −1 at the place of pathogen attack [In Arabidopsis shoots, the basal level of SA amounts to 0.25–1 µg per gram of the fresh weight, rising up to 20 µg.gn attack  (reviewen attack ). In mann attack ,34). Then attack  (reviewen attack ). The atn attack , and mutn attack ,39,40,41Scutellaria baicalensis roots [SA can also accumulate in roots in response to abiotic stresses such as aluminum, cadmium, chilling, salt, drought, UV-B radiation exposure, alkalinity, and iron- and nitrogen deficiency , echoingis roots .Scutellaria baicalensis roots also decrease when seedlings are treated with 300 µM SA [SA (50 μM) treatment promotes adventitious root development at the base of cucumber hypocotyls and strongly increases endogenous SA levels in the rooting zone . It is w00 µM SA . Therefo00 µM SA ,62. Prim00 µM SA . A low SCPR5, DND1, PI4KIIIꞵ1, PI4KIIIꞵ2 etc.) were described in relation to Arabidopsis [ddm1 mutant background.Growing evidence indicates that normal plant growth requires that optimal levels of endogenous SA are maintained. Accordingly, a number of negative regulators that alleviate SA accumulation  emergence, starting from water uptake by dry seeds (imbibition) and being completed with testa (seed coat) rupture and the radicle protrusion (reviewed in ). The prA concentration-dependent effect of SA priming on seed germination, namely, an increased germination percent at low SA concentrations and a decreased percent at higher levels, was reported in experiments on carrots, cucumbers, and wheat ,70,71,72Limonium bicolor (80–280 µM), black cumin (200–500 μM), rice (700 µM), and Bromus tomentellus (1.5–2 mM) [Lactuca sativa, Deschampsia flexuosa, and Chamaenerion angustifolium (10 mM) [For many species, SA concentrations that only activate or only inhibit germination have been described so far . SA incr.5–2 mM) ,80,81,82 (10 mM) .ABA INSENSITIVE5 (ABI5) [Other hormones also participate in primary root emergence. Only promoting effects are reported for ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and brassinosteroids, and only inhibitory effects for jasmonic (JA) and abscisic (ABA) acids and auxin ,106,107.5 (ABI5) . However5 (ABI5) ,116. GAs5 (ABI5) ,118.A concentration-dependent effect of SA on the radicle/primary root length in dicots was shown for fenugreek, cucumber, lentil, and bean ,119,120 aim1 mutant, the contrary occurs. SA deficiency results in a decreased expression of several CYCLIN genes, a reduced RAM size, and the decreased longitudinal length of mature cells [CYCLIN genes and makes the RAM size and longitudinal length of mature cells in the aim1 mutant similar to those in SA-treated wild-type plants. SA inhibits the expression of genes related to redox homeostasis and ROS scavenging to maintain the ROS accumulation necessary for RAM activity. SA provides this partially through the induction of transcriptional repressors WRKY62 and WRKY76.SA controls root growth by regulating root apical meristem (RAM) activity ,25,143. re cells . An SA teds16 mutant, which is deficient in SA biosynthesis, this occurs at a lower (5 nM) concentration.SA regulates root growth along with other plant hormones. Concentration-dependent effects on root growth were shown also for auxin , ABA, and brassinosteroids ,148,149.In Arabidopsis, predetermination of the lateral root founder cells occurs in a subset of xylem pole pericycle cells, followed by several anticlinal divisions, and culminates with cell elongation (reviewed in ). In theCatharanthus roseus hairy root line obtained from Agrobacterium rhizogenes infected leaves, SA at a very low concentration (10 fM) increases lateral root number, causes their early emergence (closer to the root tip), and enhances their growth [Adding SA 3–250 µM) to the growth medium reduces the number of lateral roots and lateral root primordia in Arabidopsis seedlings 0 µM to t,128,188 r growth . HoweverPopulus, gibberellin applied directly to the shoot apex inhibits lateral root initiation via crosstalk with auxin and ABA [In Arabidopsis, IAA promotes lateral root initiation at low concentrations (1–5 nM) and inhibits it at higher concentrations (25 nM and more) . In addi and ABA . On the  and ABA . Brassin and ABA ,169,172.The dose-dependent effects of SA on adventitious rooting is described in relation to three species: Arabidopsis, azalea, and mung beans, where low SA concentrations  increase the percentage of plants with adventitious roots or the number of adventitious roots, and higher concentrations  decrease these parameters ,142,195 In cucumber, SA (50–100 μM) competitively inhibits the formation of IAA-Asp by CsGH3.5, increases free IAA level, and promotes the formation of adventitious roots . IAA  auxin transporters in a concentration-dependent manner. Both low (30 µM) and high (150 µM) SA concentrations reduce PIN2 and PIN7 levels, while only a high SA concentration decreases PIN1 by 40%, whereas a low concentration of SA elevates PIN1 by 30% . It is wnscripts   . This paelopment ,208 and elopment , the funelopment .GH3.5/WES1 gene, encoding an auxin-conjugating enzyme (IAA-amido synthetase), have a decreased free IAA level, increased SA content, a reduced number of lateral roots [Along with the modulation of auxin transport, an SA treatment may enhance IAA content in the root. The expression of TRP AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1), the first enzyme in the main pathway of auxin biosynthesis, increases three-fold in root tips exposed to SA (both 30 µM and 150µM) . Arabidoal roots , and a ral roots . GH3.5/WDiabrotica virgifera larvae, which are soil-borne pathogens, seriously damages roots, causing an increase in IAA and SA in different parts of the maize root tip . SA’s effect on auxin signaling in roots is concentration-dependent [The complex interactions between SA and IAA differentially influence root architecture. In maize, IAA treatment (1 µM) decreases overall root biomass and root length, while SA (1.5 µM) increases both characteristics . Attacksependent . In Arabependent . Auxin aThe root stem cell niche (SCN) consists of the mitotically inactive SCN organizer and the quiescent center (QC) and is surrounded by stem cells, including cortex/endodermis initials (CEIs), stele cells initials (SCIs), epidermal/lateral root cap initials, and columella stem cells (CSCs) ,212 (revconstitutively activated cell death 1 (cad1) mutants [dnd1 and dnd2. A further increase in the SA concentration  does not affect the distal meristem organization compared to the wild type aside from the bigger size of the columella cells and the lack of starch granules.Both an SA treatment (10–30 µM) and an elevation of endogenous SA levels contribute to increases in the frequency of QC cells division, which are normally relatively mitotically inactive. Elevated SA levels give rise to additional QC cells with decreased levels of WUSCHEL RELATED HOMEOBOX 5 (WOX5), the QC marker ,215. In  mutants  and in w mutants . EnlargeThese alterations in the distal meristem occur via at least two mechanisms: changes in ROS level and in auxin distribution. SA signaling promotes ROS production and homeostasis ,215. ROSExperiments with plants treated with auxin, possessing excessive auxin accumulation and with auxin-deficient mutants, confirm the role of auxin in SA-induced columella changes. Seedlings, treated with NAA (1–5 µM) and PIN-dependent auxin transport inhibitor 1-N-naphthylphthalamic acid , as well as transgenic lines with increased endogenous auxin levels, also lose the CSC layer in their root tips due to precocious CSC differentiation . In contIt should be noted that a variety of other hormones play an important role in the control of QC cell division and CSC differentiation. Elevated levels of endogenous ethylene or an ACC treatment (50 µM) also cause extra cell divisions in QC and the formation of several additional cells with QC identity . InhibitCatharanthus roseus hairy root tissue cultures obtained from Agrobacterium rhizogenes infected leaves, SA at a very low concentration (10 fM) also expands the root cap width and increases the number of columella cells [The prolonged (3–5 days) treatment of 3–4 DAG seedlings with SA (30 µM) also enlarges the distal meristem width by disturbing cell division planes and increasing division frequency of epidermal/lateral root cap initials and CEIs . Normallla cells .A. thaliana [SA (30 µM) changes radial root patterning in the epidermis and subepidermal tissues in thaliana . Multiplthaliana ,25. Normthaliana ,222.The low SA concentration induces middle cortex formation using two aforementioned mechanisms involved in SA-induced changes in the distal meristem: SA increases auxin accumulation  and promscr mutants prematurely form the middle cortex at phloem poles (starting at 3 DAG and being extensive by 7 DAG) indicating that SCR regulates both the timing and location of middle cortex development [It is not clear if other pathways contribute to SA-induced formation of the middle cortex. Although SCARECROW (SCR) is not an SA target , scr mutelopment . SHR proelopment . In contelopment ,226,227.elopment . SEUSS ielopment . GA downelopment ,229. GA-The endodermis can produce several cortex layers while maintaining a single endodermis layer. For example, in white mustard, which belongs to the Brassicaceae family, as does Arabidopsis, there are four concentric rings of cortical cells, organized in such a way that the cells of each ring are located opposite each other, forming radial intercellular spaces from the endodermis to the epidermis, whereas in tomato, in five cortex layers, the cell positions in the concentric rings are alternate . In maizCassia tora, which is associated with an increased tolerance of seedlings to Al [Due to the sessile nature of plants, roots react to various abiotic stresses, changing the root system architecture. Treatment with SA completely removes the consequences of weak and moderate stresses and partly recovers inhibition of root growth caused by severe stresses . For exags to Al . In the SA priming of seeds (soaking seeds in an SA solution and subsequent drying) either completely or partially recover seed germination inhibited by salinity, drought, and Cd stresses ,82,236 . The effCarum copticum, 10 nM; Arabidopsis, 100 μM; and Limonium bicolor, 0.08–0.2 mM) rescue salinity-induced inhibition of seed germination, while higher doses  enhance it [+ leakage from cells due to acute salt stress, whereas high SA concentrations not only inhibit root growth but also have no impact on K+ leakage [+ leakage and H+ influx are most strongly decreased in roots treated with low SA concentrations (10–50 μM) [For several species, low SA concentrations  ,236,240 0–50 μM) .SA protection of the root system under stress conditions is also related to the optimal SA endogenous level. For example, soil alkalinity, one of the threats to crop productivity, decreases both tomato root length and dry weight . Treatmenpr1 and ein2 mutants have a lower decrease in root fresh weight compared to the wild type and npr1 ein2 double mutant. SA-mediated stress responses may recruit other hormonal pathways. In barley exposed to Cd (15 µM), IAA content in root tips increases three-fold [In roots, the alleviation of abiotic stress damage with SA occurs via SA crosstalk with other hormones and via hydrogen peroxide and nitric oxide produced in response to both SA and stress ,243,244 ree-fold . An incrree-fold ,246.Fusarium oxysporum-induced cell wall reinforcement in the root due to enhanced lignin synthesis, thereby alleviating pathogen propagation [Pseudomonas syringae strain Pto DC3000 invades Arabidopsis plants through emerged lateral roots and then induces lateral root formation by producing auxin, which activates the ARF7/ARF19–LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16)/LBD18 regulatory module [PATHOGENESIS-RELATED GENE 1 (PR1) and PR2 transcription, thereby decreasing the number of potential pathogen entry sites [PR1 and PR2 to derepress lateral root development. In response to pathogen attacks, plants accumulate SA, which represses auxin signaling [Due to their direct contact with the soil, roots are vulnerable to soil-borne pathogens such as fungi, bacteria, viruses, nematodes, and herbivorous insects. Recent studies demonstrate that some SA-induced morphological and morphogenetic changes are a part of the strategy that SA utilizes to restrict pathogen invasion in the root. Some pathogens, for example, viruses, are capable of invading epidermal and cortical cells intracellularly through plasmodesmata ,248,249.pagation . SA antay module ,252. SA ry sites . Notablyignaling  and tranignaling .In addition to pathogens, plant roots contact a plethora of non-pathogenic soil microorganisms. The microbial community associated with the plant roots facilitates physiological and morphological functions of roots, including organogenesis and root architecture NahG, promote infection thread and nodulation, including the determinate-nodule-type plant Lotus japonicus [L. japonicus [In legumes, symbiotic Rhizobium bacteria under nitrogen-limiting conditions, trigger the plant-guided formation of novel root organs called nodules, which promote rhizobia-mediated nitrogen fixation from the atmosphere . Thaponicus . The bacaponicus ,262,263.aponicus . This giaponicus .Serratia marcescens, strain 90–166. It is alleviated in SA-deficient plants overexpressing NahG, highlighting the impact of the SA signaling pathway in this morphological trait [Another example of SA participation in symbiont-induced morphogenesis is the induction of second-order lateral root development in Arabidopsis, elicited by the rhizobacterium al trait .Salicylic acid is often considered in the context of its protective mechanisms against biotic and abiotic stress. Providing this dual function, SA greatly influences root development starting at seed germination, through to root elongation, root branching, and adventitious rooting. Treatment with SA often causes dwarf plant phenotypes, which is interpreted as being an SA-induced shift of plant resources from growth to defense. To ensure optimal plant growth in an unfavorable environment, different mechanisms controlling the growth-defense balance in plants have evolved, in which SA plays an important role. First, SA-mediated concentration-dependent positive/negative feedback regulation of its own biosynthesis fine-tunes this balance . AnotherOn closer inspection, SA-mediated morphological changes such as the reduction of lateral root formation, enhanced adventitious rooting, nodulation, cell wall lignification, and plasmodesmal closure, can directly impede pathogen invasion in the root or facilitate plant growth under adverse conditions. In this context, it is important to highlight that most of the SA effects are concentration-dependent, which was demonstrated at least in several species. Consequently, there are two bioactive concentration windows for SA in the root system; at low levels, it acts as a developmental regulator, and at high levels, SA acts as a stress hormone . It has


Headache and facial pain  were the most common symptoms. The most involved sinonasal site was the maxillary sinus . The overall survival rate at the three-month follow-up was 79.9%. COVID-19-related aggressive inflammatory response, uncontrolled glycemic level, and rampant use of steroids are the most important predisposing factors in developing COVID-19-associated invasive fungal sinusitis.This study aimed to determine the patient demographics, risk factors, which include comorbidities, medications used to treat COVID-19, and presenting symptoms and signs, and the management outcome of COVID-19-associated invasive fungal sinusitis. A retrospective, propensity score-matched, comparative study was conducted at a tertiary care center, involving 124 patients with invasive fungal sinusitis admitted between April 2021 and September 2021, suffering from or having a history of COVID-19 infection. Among the 124 patients, 87 were male, and 37 were female. A total of 72.6% of patients received steroids, while 73.4% received antibiotics, and 55.6% received oxygen during COVID-19 management. The most common comorbidities were diabetes mellitus (83.9%) and hypertension (30.6%). A total of 92.2% had mucor, 16.9% had aspergillus, 12.9% had both, and one patient had hyalohyphomycosis on fungal smear and culture. The comparative study showed the significant role of serum ferritin, glycemic control, steroid use, and duration in COVID-19-associated invasive fungal disease ( Coronavirus disease 2019 (COVID-19) is an infection caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Since the first case was detected in December 2019 in Wuhan, China, there have been various twists and turns in the understanding of its pathophysiology, diagnosis, management, sequelae, and complications ,4,5.Clinically, invasive fungal sinusitis can present with atypical signs and symptoms similar to complicated sinusitis ,7. InvasFurthermore, COVID-19 possibly plays an essential role because affected patients show overexpression of inflammatory cytokines and impaired cell-mediated immunity with a decreased cluster of differentiation of 4 and 8 positive T-helper (CD4+ T and CD8+ T) cell counts. This indicates susceptibility to fungal coinfection ,11, alonA retrospective, hospital-based, observational study was conducted at SGPGIMS, Lucknow Tertiary Referral Center, India, over six months, from April 2021 to September 2021. All patients with invasive fungal disease of the paranasal sinuses, either COVID-19-positive or recovered from COVID-19 infection, were included in the study. A control group was formed by collecting data on COVID-19 patients admitted at our center during the same duration. From these control patients, 124 propensity score-matched  patients were identified among those who did not develop invasive fungal sinusitis. Patient age, gender, history, COVID-19 management details, clinical presentation, imaging findings, comorbidities, pathology, and follow-up information was obtained, recorded, and analyzed.Statistical Analysis: Continuous variables were represented as mean ± standard deviation and median and interquartile range and categorical variables in frequency (%). The Mann–Whitney U-test was used to compare medians, whereas, for proportions, the Chi-square test was used between the two groups. Propensity score matching was performed in COVID-19 patients with and without invasive fungal sinusitis, where age, sex, type of respiratory support , and comorbidities (hypertension and diabetes) were matched. Binary logistic regression analysis was used to assess the independent predictors (from treatment and inflammatory variables) of the invasive fungal sinusitis compared with COVID-19 patients who did not develop invasive fungal sinusitis. A value of p < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 23.0 software .A total of 124 patients were diagnosed with invasive fungal sinusitis with a history of COVID-19 infection; of them, 87 were male, and 37 were female. The mean age of patients was 51.7 ± 11.54 years (range: 28–82). Twenty patients were COVID-19 positive at the time of presentation, and the remaining 104 had been documented with infection earlier and had recovered. Among the 124 patients, only 66 (53.2%) had been admitted to the hospital for treatment of COVID-19, while the remaining 58 (46.8%) were in home isolation. During management of COVID-19, 90 (72.6%) patients received corticosteroids for a mean duration 11.3 ± 6.6 days  received antibiotics (48 (52.7%) intravenous antibiotics and 43 (47.3%) oral antibiotics), and 69 (55.6%) were on oxygen support ; NRBM, 9 (13%); BIPAP, 9 (13%); mechanical ventilation, 7 (10.1%)) for a mean duration of 9.5 ± 8.13 days (range: 0–45). Tocilizumab was used in 3 (2.4%) patients. The mean duration between becoming COVID-19 positive and the appearance of the first symptom of invasive fungal sinusitis was 35.4 ± 37.15 days (range: 6–292). Fungal sinusitis confirmation was performed by KOH mount, fungal culture, and histopathological examination. On evaluating the type of fungi, we found that 92.1% had mucor, 16.9% had aspergillus, 12.9% had both, and one patient (0.8%) had hyalohyphomycosis. Serum ferritin  was elevated in 78.3% (97/124) of patients, with a mean value of 1780.3 ± 7885.63 .The most commonly associated comorbidity was diabetes mellitus (DM) (83.9%). Among the 104 patients diagnosed with diabetes, 9 (8.6%) had a history of a recent onset, probably caused by corticosteroid therapy given during COVID-19 treatment. Of the 104 patients, 88 (84.6) had uncontrolled DM, and 16 (15.4) had controlled DM based on HbA1c values. The mean HbA1c was 9.1 ± 2.26. Other comorbidities in our study populations were hypertension seen in 38 (30.6%), heart disease in 8 (6.5%), liver disease in 5 (4%), hypothyroidism in 5 (4%), and kidney disease among 16 (12.9%) and 5 (4%) was post-renal transplant patients on immunosuppressive drugs. One patient was HBsAg positive, one was HCV positive, and one had acute chronic pancreatitis. Three patients had concomitant pulmonary and rhino-orbital mucormycosis .The “classic” triad of DM, steroid therapy, and oxygen use was present in 54 (43.5%) patients, while 29 (23.4%) had only DM, and 5 (4%) received only steroids. No patient developed invasive fungal sinusitis with oxygen treatment alone. Four patients were nondiabetic and did not use steroids and oxygen during treatment of COVID-19. Of these four, one patient was a post-renal transplant and was on immunosuppressant therapy, and the other three did not have any comorbidities.p < 0.001), HbA1c , proportion of patients receiving steroid , and duration of steroid use  were significantly higher in invasive fungal sinusitis patients compared to control patients. Multivariate binary logistic regression was used to calculate the adjusted odds ratio (AOR) after including significant variables from univariate analysis. In this analysis, except the use of steroids, the other variables, i.e., serum ferritin , HbA1c , and duration of steroid use , were significant and found to be independent risk factors in development of invasive fungal sinusitis  with those who did not develop mucor. Comparative univariate analysis showed that serum ferritin  patients, followed by ethmoid sinuses in 87 (70.2%) patients. The lateral nasal wall was involved in 77 (62.1%) patients, and the sphenoid sinus was involved in 56 (45.2%) patients. Frontal sinus involvement was less common, seen only in 25 (20.2%) patients. Pterygopalatine fossa and infratemporal fossa were involved in 46 (37.1%) and 38 (30.6%) cases, respectively. Orbital involvement was seen in 44 (35.5%) cases, and the palate was involved in 41 (33.1%) cases, while 23 (18.54%) patients showed intracranial extension, with the spectrum containing five frontal lobe abscesses, three temporal lobe abscesses, two parietal lobe abscesses, nine cavernous sinus thrombosis, three internal carotid artery (ICA) thrombosis, and three fungal cerebritis. Nineteen patients had skull base osteomyelitis .Among the 124 patients, 110 were treated with combined antifungal therapy and surgery, while 14 patients were on antifungal therapy alone without surgical debridement. The reasons for avoiding surgery in these 14 patients were that 2 patients did not consent to surgery, 10 patients were COVID-19 positive (risks outweighed benefits), while the other two were admitted to ICU with severe sepsis, pneumonia, kidney failure, and overall poor general condition, making surgical intervention too unreasonable a risk. Among the 110 patients undergoing surgery, 93 were primary cases, and 17 were revisions. Of the 17 patients, 14 underwent revision surgery, and only antifungals were used to manage 3 patients. Only one patient underwent surgery in the RT-PCR-positive status; the rest underwent surgery after two consecutive negative COVID-19 RT-PCR reports. Orbital exenteration was performed in 5 patients. Intracranial abscess drainage was performed in 5 patients.Amphotericin B  was given as the primary antifungal drug in 116 patients, and isavuconazole was given in 8 patients. Posaconazole and voriconazole were used as step-down therapy for three months according to histopathology report and microscopy report.The overall survival rate was 79.8% (99/124) in our study after a mean follow-up of three months. Of the 25 (19.4%) patients who expired, 11 had an active COVID-19 infection at the time of death. Finally, 13 died due to severe respiratory failure and sepsis due to COVID-19, 9 had intracranial extension , and 2 patients had simultaneous pulmonary mucormycosis. Three patients were post-renal transplants and were on triple immunosuppressants. Four patients developed hemiparesis in the immediate post-op period.Mucormycosis or zygomycosis, also called phycomycosis, initially described in 1885 by Paltauf, is an uncommon and aggressive fungal infection that usually affects patients with alteration of their immunological system . AlthougIn our study population, the mean age of COVID-19 patients admitted to the hospital was 48.24 years (range: 36–61), and 71.8% of patients were males . The medThe etiology of COVID-19-associated invasive fungal sinusitis appears multifactorial. In our population with COVID-19, DM was seen in 31.7% of hospitalized patients. New-onset DM was seen in 20.6% of patients with mild to moderate COVID-19 ,22. HypeIn the studies of Kursun et al. , MohammaExtensive use of steroids and broad-spectrum antibiotics in the management of COVID-19 leads to the development or exacerbation of a pre-existing fungal disease . AccordiThe mode of contamination occurs through the inhalation of fungal spores. Our data of COVID-19-associated invasive fungal sinusitis show that 55.6% (69 of 124) patients required oxygen support during the treatment of COVID-19, but, at the same time, either they had DM or had received corticosteroids in some form. No patient in our study who received only oxygen therapy as a treatment modality for COVID-19 developed invasive fungal sinusitis, suggesting that contaminated oxygen is unlikely to be a cause of infection. In addition, four (3.2%) patients were nondiabetic and did not receive oxygen and steroid; three (75%) out of four were immunocompetent but still developed mucormycosis, raising a possible role of COVID-19 as a culprit. Further, an ubiquitous, fungi-like mucor, coupled with poor hygienic practices followed at the individual and community levels, can be speculated as one of the causes for such high incidence .Invasive fungal sinusitis occurs both concomitantly with COVID-19, as well as post-recovery. In a large multicenter study of 2285 patients by Sen et al. , 56% of In our study, the mean duration between COVID-19-positive RT-PCR and the first symptom of invasive fungal sinusitis was 35.4 ± 37.15 days (range: 6–292). In our study, 4.03% developed signs and symptoms in 7 days, 22.58% developed between 8 and 15 days, 32.2% developed between 16 and 30 days, and 33.06% developed between 30 and 90 days from the diagnosis of COVID-19. Only 8.06% developed symptoms after 90 days. This revealed sinusitis as a red flag sign in the immediate post-COVID-19 period, specifically in high-risk patients. This proposed need for early ENT examination and emphasized the education of patients and families with a checklist of symptoms of invasive fungal sinusitis because 92% of patients developed symptoms within three months after recovery from COVID-19. It has been observed that patients with early diagnosis and limited disease extension have the best outcome with minimal morbidity and mortality [Clinically, invasive fungal sinusitis can present with atypical signs and symptoms similar to complicated sinusitis, such as headache, nasal blockage, nasal crusting, proptosis, facial pain and edema, ptosis, chemosis, and even ophthalmoplegia, with headache and fever and various neurological signs and symptoms such as lower cranial nerve palsy altering sensorium in patients with intracranial extension ,7. In ouA study by El-Kholy et al. showed that the most involved sinonasal site was the lateral nasal wall (86.1%), with the ethmoid (72.2%) and sphenoid (55.6%) sinuses being next. In this study, 27.8% showed extension intracranially, and 33.3% showed palatal involvement . A largeAs soon as the patient presented with symptoms suggestive of invasive fungal sinusitis, our protocol was to get a fresh contrast-enhanced MRI and CECT of the nose and PNS to know the extent of the disease and for surgical planning. We also performed a diagnostic nasal endoscopy and sent nasal swabs for fungal microscopy and culture. The MRI shows better soft-tissue delineation and better characteristic findings, but the CT provides better bony details. Diagnostic nasal endoscopy and nasal swabs were not always helpful, but they could be performed by the bedside and without any delay when needed. Complete blood count (CBC), liver function test (LFT), renal function test (RFT), blood coagulation profile, HbA1c, an inflammatory marker, COVID-19 RT-PCR test, blood sugar charting, and all investigations for surgical fitness were sent immediately. Management for high blood sugars was started, and after checking RFT, we started antifungals accordingly with strict monitoring. Repeat RFT, LFT, and serum electrolyte were performed daily, and medications were tailored to any changes. As soon as the patient was considered fit for surgery, they were taken for debridement, and antifungals were continued in the post-op period according to the extent of involvement of the disease.Medical management was with liposomal amphotericin B in 5–10 mg/kg/day doses up to a total cumulative dose of 3 to 5 g depending on the extent of the disease and patient tolerance. If the patient had a hypersensitivity reaction or RFT was deranged, 200 mg isavuconazole TDS was given for two days, followed by 200 mg once daily. For patients with orbital involvement with intact vision or who did not give consent to orbital exenteration, multiple transcutaneous retrobulbar amphotericin B (TRAMB) injections were given. Posaconazole was used in step-down therapy in a dose of 300 mg BD for two days, followed by 300 mg OD for three months with monitoring of LFT.Surgical management was performed by a multidisciplinary team, including otorhinolaryngology, ophthalmology, neurosurgery, oral maxillofacial surgery, and plastic surgery teams, depending on the requirement for the case. Our approach was purely endoscopic in most cases. Endoscopic modified Denker’s removal of the middle and inferior turbinate, wide middle meatus antrostomy, ethmoidectomy, sphenoidotomy, frontal sinusotomy, medial/inferior partial maxillectomy, orbital decompression by removal of medial and inferior wall, clearance of ITF and PPF, orbital exenteration, craniotomy, and removal of the abscess were included as part of the surgical approach depending on the needs of the case.During follow-up, nasal endoscopy and suction clearance were performed every 15 days. Follow-up MRI was performed after 1 and 3 months of posaconazole course or earlier if patients developed any new symptoms. Two patients underwent revision surgery. One developed palatal necrosis, and the other developed a lacrimal abscess. Patients are still under close follow-up for any symptoms and blood sugar monitoring.The prognosis of invasive fungal sinusitis is poor, with reported mortality rates ranging from 33.3% to close to 100% in disseminated infections even if aggressive debridement and intravenous antifungal agents are utilized ,33. OrbiThis study is associated with certain drawbacks. First, it was a retrospective analysis. A prospective study with this aspect could reveal more about the epidemiology and risk factors leading to this condition. Secondly, this study was hospital-based in a tertiary referral center, where most cases were referred from primary and secondary treatment centers from all over the state.COVID-19 patients, especially high-risk ones, must be followed up by ENT after recovery. COVID-19-related aggressive inflammatory response, uncontrolled glycemic level, and rampant use of steroids are the most important predisposing factors in developing COVID-19-associated invasive fungal sinusitis. A high index of clinical suspicion, diagnosis at an early stage, inflammatory marker monitoring, strict glycemic control, aggressive surgical debridement, and use of antifungal agents are essential for a successful outcome. Finally, to prevent such a dangerous and costly disease, stricter policies have to be implemented and followed to prevent injudicious antibiotic and steroid use. Over-the-counter sale of these drugs should be prohibited, and doctors should strive to follow national and internationally set protocols for their use.


According to the data analysis from recent studies in the past 20 years, nicotine exerts much more anti-inflammatory effects than pro-inflammatory ones, especially in ulcerative colitis, arthritis, sepsis, and endotoxemia. On the other hand, in oral inflammation, nicotine promotes and aggravates some diseases such as periodontitis and gingivitis, especially when there are harmful microorganisms in the oral cavity. We also carefully analyzed the nicotine dosage to determine its safe and effective range. Furthermore, we summarized the molecular mechanism of nicotine in these inflammatory diseases through regulating immune cells, immune factors, and the vagus and acetylcholinergic anti-inflammatory pathways. By balancing the “beneficial” and “harmful” effects of nicotine, it is meaningful to explore the effective medical value of nicotine and open up new horizons for remedying acute and chronic inflammation in humans.As an anti-inflammatory alkaloid, nicotine plays dual roles in treating diseases. Here we reviewed the anti-inflammatory and pro-inflammatory effects of nicotine on inflammatory diseases, including inflammatory bowel disease, arthritis, multiple sclerosis, sepsis, endotoxemia, myocarditis, oral/skin/muscle inflammation,  Nicotine, also called 3-(1-methyl-2-pyrrolidinyl) pyridine, normally accounts for about 5% (w/w) of the tobacco plant and is the major psychoactive and addictive component in tobacco smoke. It belongs to alkaloids, with a pyridine cycle and a pyrrolidine cycle, and occupies a higher proportion of ~95% of the total alkaloids in tobacco leaves  is a global problem. It impacts all aspects of life and affects people of all ages, most commonly during adolescence and young adulthood . It usuaIn general, nicotine is beneficial in ulcerative colitis; in particular, nicotine transdermal patches or nicotine enemas have shown significantly improved histological and global clinical scores of colitis, inhibited pro-inflammatory cytokines in macrophages, and induced protective autophagy to maintain intestinal barrier integrity , 28. In via the drinking water suppressed the mucosal vascular address in cell adhesion molecule‐1 (MAdCAM‐1) expression on the endothelium of colonic microvessels and endothelial bEnd.3 cells and the recruitment of leukocytes to the inflamed colonic microvessels, which is the underlying pathophysiology of DSS‐induced colitis  neurons and the visceral hypersensitivity of the number of abdominal constrictions in response to an intraperitoneal injection of 0.6% acetic acid in DSS-treated male C57BL/6J mice, finally relieving acute DSS colitis  in the drinking water of a5-deficient mice for 7 days before the induction still significantly ameliorated the colonic inflammation course by reducing prostaglandin E2 (PGE2) and MPO, indicating that other nicotinic acetylcholine receptor subunits also mediate the anti-inflammatory effects of nicotine (1–L2) neurons in vitro (in vivo (bath-applied) -induced mouse or rat model, with body weight loss, colon shortening, missing colonic mucosal epithelial cell, gland integrity loss, inflammatory cell infiltration, colonic tissue injury, and typical inflammatory reaction. In DSS-induced male C57BL/6J mice, nicotine (0.1 mg/ml) administration  colitis . Oral ni colitis . Nicotinin level . Further colitis . In fema colitis . It seemn 7 days . In malen 7 days  also founicotine . α7 nAchin vitro  and in vapplied) . Neurona4) formation by downregulating the colonic IL-1β and monocyte chemoattractant protein (MCP)-1 protein expression in the inflamed colon of animals with colitis  in the isolated colonic segments induced by toxin A dose-dependently inhibited the harmful effects, but not the ileum experiment. Furthermore, nicotinic inhibition includes the downregulation of transient receptor potential vanilloid subtype 1, LTB4, and pro-inflammatory neuropeptide (substance P) via nAChRs, but not directly -induced colitis in Sprague–Dawley rats, subcutaneous nicotine (a single dose of 8 mg/kg or triple doses of 4 mg/kg nicotine) reduced the colonic lesion size, MPO activity, luminol-amplified free radical generation, and leukotriene B4  30 min after a severe burn injury, which improved the gut barrier function of intestinal permeability by preventing the decreased expression of occludin and ZO-1 and maintaining their localization. In addition, nicotine was further shown to improve intestinal inflammation by inhibiting the stimulation of IFN-γ, TNF-α, and IL-1β in intestinal epithelial cell CaCO-2 cocultured with enteric glial cells . HoweverIn small intestinal ulceration induced by indomethacin in male C57BL/6 mice, nicotine  reduced the severity of intestinal lesions by up to 53.3, 74.7, and 81.3%, respectively, in a dose-dependent manner by suppressing the MPO activity and iNOS expression due to the inhibition of NFκB. This effect was impaired by methyllycaconitine, a selective antagonist of α7 nAChR antagonist .via their connectivity with enteric nerves expressing α7 nAChRs  significantly inhibited the lipopolysaccharide (LPS)-induced mRNA expression of IL-1β and iNOS, but not TNF-α and MCP-1, in primary male Sprague–Dawley rats’ mesothelial cells 7 nAChRs .4. This effect was similar to 375 μg/ml sulphasalazine but greater than 100 μg/ml nicotine, indicating that low doses of nicotine were more effective in reducing inflammation than higher doses  reduced macroscopic damage with a reduction in MPO, iNOS, TNFα, and LTBer doses . In Croher doses .In a human experiment, in 30 patients with left-sided ulcerative colitis refractory to rectal mesalamine alone, by calculating the Rachmilewitzis activity index confirmed by endoscopic and histological findings, transdermal nicotine (15 mg) plus mesalamine enemas (4 g) showed significantly therapeutic success in 4 weeks than mesalamine tablets  plus mesalamine enemas (4 g) .via vagus nerve activation, with no apparent adverse events -infected macrophages, providing important insights to understand the effect of nicotine on worsening inflammation symptoms in patients with Crohn’s disease , arthritis is any disorder that affects the joints, including joint pain and stiffness, usually accompanied by redness, warmth, swelling, and decreased range of motion of the affected joints, which typically worsens with age. There are >100 types of arthritis. The most common types of arthritis are rheumatoid arthritis and osteoarthritis . Nicotin+IL-17A+Th17 cells expressing α7 nAChR stimulated by nicotine may decrease their pro-inflammatory function  suppressed the arthritis index of hind paw swelling and weight gaining and decreased some hematological and biochemical parameters, including rheumatoid factor, C-reactive protein, NO, MPO, IL-1, and IL-17. Furthermore, the combination of 1.25 mg/kg nicotine and 50 mg/kg thymol  resulted in a synergistic benefit and tolerable safety margin in alleviating this rheumatoid arthritis model  reduced mechanical allodynia, cartilage degradation, and the upregulation of MMP9 production by macrophages  pathway . In mono7 nAChRs . With th7 nAChRs . In surgc system . In a marthritis .M. tuberculosis H37Ra (Mtb)-induced arthritis of Lewis rat models, pretreatment with nicotine  before induction of arthritis exacerbated the disease. In contrast, post-injection of nicotine after the onset of arthritis decreased the disease severity. Thus, the critical variables in the opposite outcomes, including the timing of nicotine administration and the induction model, differed quantitatively and/or qualitatively from that of male rats. In female ovariectomized BALB/C mice immunized with chicken collagen II in complete Freud’s adjuvant injected in the tail root, nicotine (0.03%) in drinking water caused arthritis by supporting the non-exhausted PD-1-IL-7R+CD8+ T cells, possibly by inducing the release of survivin protein from the bone marrow (−8 to 10−7 M) in a high concentration of MMP1/MMP13 and two proposed markers of osteoarthritis, fibronectin (FINC) and chitinase 3-like protein 1 (CHI3L1), in primary chondrocytes from patients also reminded us of the cautious and judicious use of nicotine in osteoarthritis treatment , for a short time (24 h), could exert a suppressive effect on the production of inflammatory mediators, including IL-8 and ICAM-1 induced by LPS or TNF-α, indicating that nicotine may serve as an anti-inflammatory agent. However, nicotine also inhibited β-defensin induced by Porphyromonas gingivalis LPS, indicating that it may allow the overgrowth and invasion of potential periodontal pathogens. Importantly, nicotine did not affect HSC-2 proliferation/viability in this concentration range. It is impossible to ignore that nicotine may have a strong toxic effect on different cell types at concentrations higher than 10−3 M  exhibited decreased levels of bone alkaline phosphatase and osteocalcin as important markers for bone mineralization and activity of osteoblasts, with increased TNF-α and COX−2 expression levels causing ischemia and inflammation of the periodontium and higher alveolar bone loss. Here catalpol was shown to improve nicotine-induced injury to the alveolar bone by promoting alveolar bone mineralization and inhibiting the inflammatory factors (in vivo study on periodontitis-susceptible Fischer 344 rats injected with nicotine in the neck skin (0.8 mg/kg for 3 weeks) and intraperitoneally injected with LPS (100 μg/kg) found that these developed significantly more periodontal bone loss and periodontal tissue destruction, while nAChR antagonist mecamylamine abolished this effect. Nicotine treatment alone indirectly reduced TNF-α, TGFβ, and IL-10 production, indicating that the acetylcholine receptors activated by nicotine may suppress protective immune responses through the cholinergic anti-inflammatory pathway by altering the immune system responses and the susceptibility to periodontitis  and the NF-κB/c-Fos/NFATc1 signaling axis, involved in inflammatory bone destruction and osteoclast differentiation, with an increase in phospholipase D (PLD) 1 and PLD2 isoforms  dramatically repressed cell viability and increased apoptosis, which was associated with the collapse of periodontal tissues and elevated matrix metalloproteinases MMP1, MMP3, and cytokines IL-1β, IL-6, IL-17, IL-21, and chemokines CXCL12. The receptor α7 nAChR played a major mediating role, and its inhibitor α-bungarotoxin reversed nicotine-induced apoptosis, increased viability, and attenuated the nicotine-triggered production of these factors -releasing molecule-3 pretreatment suppressed the inflammatory and osteoclastogenic cytokines, including PGE2, COX-2, and RANKL/OPG ratio by releasing CO (−9 M) alone did not affect the osteoblastogenesis/osteoclastogenesis balance of PDLSCs. However, it significantly exacerbated the destructive effect of inflammatory factors (5 ng/ml IL-1β and/or 10 ng/ml TNF-α) by decreasing the osteogenic differentiation indicators ALP, RUNX2, BSP, and OCN, increasing the osteoclast-forming indicators RANKL/OPG ratio by suppressing the expression of phosphorylated GSK-3β, and diminishing the function of its downstream factor α7 nAChR. The apoptotic effect exerted by nicotine on these first-line defensive cells, such as polymorphonuclear cells (PMNs) and mononuclear cells, may be an important feature of the pathogenesis of the periodontal disease. A brief exposure of PMN to nicotine concentrations ranging from 0.01 to 0.3% shortened the accelerated cell apoptosis in a dose-dependent manner. Concerning mononuclear leukocytes, nicotine could not induce apoptotic modifications on cells observed up to 72 h of culture time. However, it inhibited IL-1β release and procoagulant activity expression, indicating that when mononuclear cells become the predominant population in inflamed tissues, nicotine could offer an interesting exogenous example of the phlogistic balance that governs the evolution and resolution of the persistent inflammation in periodontal disease , the release of IL-1α and IL-8 cytokines increased significantly. These findings support an interactive effect of smoking and bacterial factors on host response in periodontal disease  potentiated capsaicin-evoked immunoreactive calcitonin gene-related peptide release in a concentration-dependent manner by α3, α4, or α6 nAChR subunit in trigeminal ganglia which co-localized with CGRP-/VR1 (the capsaicin receptor) in adult male Sprague–Dawley rats. These findings suggest that nicotine may predispose the buccal mucosa to an enhanced neurogenic inflammatory response to different stimulatory factors, such as thermal, chemical, and mechanical tissue damage or infection  did not influence cell viability but stimulated the concentrations of periodontal inflammation and oral pain correlation factor substance P (SP), a major mediator of neurogenic inflammation, and IL-1β. Here neutral endopeptidase (NEP) exerted a good relieving effect by downregulating the SP and IL-1β levels by cleavage . Nicotinnfection . One in ectively . This suectively .Streptococcus mutans and Candida albicans in denture biofilms. Furthermore, 4 mg/ml nicotine in tryptic soy broth significantly increased the coaggregation of these two microorganisms after 24 h of incubation, increasing the likelihood of denture stomatitis and caries  should be explored and clarified.To sum up, the 5-mM concentration of nicotine has mostly been used in many previous studies. The range of nicotine concentrations measured in the saliva of long-term snuff users is 0.6–9.6 mM . From thThe evidence on smoking and skin disease is conflicting. However, the potential for nicotine treatment is in skin conditions that occur less often in smokers than in non-smokers.An interventional study early in 1997 showed that nicotine patches suppressed the cutaneous inflammatory response to sodium lauryl sulfate and ultraviolet radiation and transiently suppressed reactive hyperemia following arterial occlusion in 10 lifelong non-smokers .In Bechet’s disease, nicotine maintained a specific anti-inflammatory effect on keratinocytes  and endothelial cells (HMEC-1) in the serum of patients. After keratinocytes and HMEC-1 were incubated with the serum of 20 patients with Behçet’s disease and 20 healthy controls for 4 h, nicotine (6 μM) significantly decreased IL-8 and IL-6 released by HMEC-1 maintained in both patients’ and controls’ sera. However, only IL-6 released by keratinocytes was maintained in patients’ sera. VEGF released by both cells increased markedly after nicotine treatment in either serum. Thus, it is likely that nicotine could suppress neutrophil-mediated inflammatory actions .In the passive cutaneous Arthus reaction, an acute inflammatory response resulting from the deposition of immune complexes in the tissue, the plasma extravasation, and polymorphonuclear leukocyte infiltration serve as indexes to evaluate the anti-inflammatory activities. Kubo et al. , 103 rep-8 M and 10-4 M, three injections of 20 μl  around the wounds once daily for 3 days] injected into full-thickness excisional skin wounds of female C57BL/6 mice minimally affected the inflammatory mediators like TNF, IL-6, and IL-12. However, it downregulated the expression of growth factors like VEGF, PDGF, TGF-β1, and TGF-β2 and the anti-inflammatory cytokine IL-10, reflecting the overall detrimental effects of tobacco on wound healing and general skin diseases (Nicotine  mice ] mice . NotablyNonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease, was alleviated by nicotine due to its anti-inflammatory effect. Tang’s group has carried out more research in this field –153. In The diseases listed in this part have not been studied very much, and mostly, the anti-inflammatory effects of nicotine have been shown. However, it has guiding significance for future research.Systemic lupus erythematosus is an autoimmune disease characterized by aberrant immune function, increased renal inflammation, and hypertension. The cholinergic anti-inflammatory pathway is impaired in systemic lupus erythematosus because of autonomic neuropathy. However, nicotine stimulation  could lower the blood pressure and proteinuria in female NZBWF1/J mice with systemic lupus erythematosus, a novel and appropriate disease model to study the link between chronic inflammation and the pathogenesis of hypertension. Additionally, chronic nicotine exposure inhibited splenic TNF-α and renal cortical MCP-1 and increased medullary IL-10. Thus, nicotine also regulated chronic renal inflammation through the immune and nervous systems acting together .Uveitis frequently leads to severe vision loss and blindness by disrupting the blood–ocular barrier and neighboring tissues associated with intraocular proliferation. In LPS-induced Wistar rats of uveitis models, nicotine  significantly reduced the protein, but not mRNA, levels of IL-6, CINC-1, and MCP-1, in serum and the levels of IL-6, IL-1β, CINC-1, TNFα, and MCP-1 in aqueous humor through α7 nAChR, suggesting the potential clinical translation of nicotine and other cholinergic agonists in uveitis .+nAChRα7+ macrophage activation in mdx skeletal muscles, reduced the inflammatory infiltrates, and increased myofiber regeneration without increasing collagen deposition or fibrosis  downregulated the activity of pro-inflammatory mediators MMP9, NFκB, and TNFα through acetylcholine secretion and F4/80fibrosis . In addifibrosis . HO-1 anfibrosis .Infectious or immunologic inflammation are important contributors to fetal growth restriction. Nicotine suppressed placental inflammation to protect the fetus in studies by the Liu group –162. In Nicotine is a well-known parasympathomimetic alkaloid, typically detected at a high level in tobacco leaves. The experimental studies reviewed here have suggested that the effect of pure nicotine monomer is not detrimental as cigarette smoke. Unlike pure nicotine, tobacco smoke contains toxic and carcinogenic substances. Although cigarette smoking has been defined as a global public health threat responsible for several diseases that can be partially prevented by quitting, smokers are less prone to some chronic inflammatory diseases. As yet, the dose and duration of nicotine treatment for these conditions is unclear. The discrepancy between the effects of nicotine on many inflammatory diseases might be attributed to the different pathophysiological mechanisms in these different models, the determined tissue, the treatment duration, or animal species (in vivo in mice (after uniform dose conversion in rats), the beneficial concentration range of nicotine in water is 6–200 μg/ml. The water intake of adult mice (~20 g) is generally 4–7 ml/day; after adjustment, the concentration is approximately 1–200 mg/kg. The concentration ranges of intraperitoneal injection, subcutaneous injection, and mini-pump infusion are 0.04–60, 0.3–48, and 1.2–24 mg/kg, respectively. After the conversion of effective utilization between different treatment methods, if all is injected intraperitoneally, the concentration range of nicotine is 0.04–600 mg/kg/day. Notably, the animal data on intraperitoneal injection are more comprehensive, and the effective dose is concentrated at 400 μg/kg/day (15 reports). A high dose is generally appropriate for acute and severe inflammation, such as sepsis  regulating oxidative stress balance through HO-1/MPO, SOD, and DAO/MDA, (2) regulating metal matrix enzyme pathways MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP14, and MMP28 and TIMP-1 and TIMP-2, and (3) regulating cell phosphorylation signaling pathways AMPK/mTOR/p70S6K/LC3II/LC3I, p62, beclin-1, IL-6/Stat3/miR-21, TLR2/MyDD88/IL-8, JAK2-STAT3-IL-6/MCP1, PI3K/Akt/iκB/NF-κB, p38/Erk/JNK/NF-κB, PI3K/MAPK /NF-κB, AP-1, NF-κB/c-Fos/NFATc1, HO-1/STAT3/tristetraprolin/TNFα, and HSP70/TNFα/iNOS. There are also specific target studies, including pro-inflammatory neuropeptide (substance P), occludin, ZO-1, RORγτ, GATA3, SOCS3, anti-cyclic citrullinated peptide (anti-CCP) antibodies, survivin, ECM molecules , PIN1, NEP, PU.1, cytokine-induced neutrophil chemoattractant (CINC)-3, and thrombin–antithrombin complexes, ARG1, NOD2, and VEGF. These factors form the signaling network of nicotine regulating inflammation  inflammatory cytokine production through signaling pathways, such as STAT3/NF-κB, in many tissues. Nicotine not only acts on the vagus nerve but also on its hepatic or splenic branches to regulate acetylcholine receptors. Recent studies showed that, in addition to regulating the circulatory, respiratory, and digestive systems, the vagus nerve also regulates the immune system. On this basis, nicotine regulated immune cells, including CD25ammation Figure 3Notably, many nicotine-induced effects in humans and animals are mediated through nicotinic acetylcholine receptors. nAChR α7 and other subtypes, such as nAChR α3, α4, α5, α6, α4β2, and α3β4, are generally activated by nicotine and expressed by numerous non-neuronal cell types, including distinct populations of astrocytes, epithelial cells, adipocytes, lymphocytes, macrophages, and keratinocytes, supporting the extensive anti-inflammatory effects of nicotine in many inflammatory diseases, even in the absence of parasympathetic innervation , 164. AmThe potential therapeutic use of nicotine is influenced by its complex pharmacology, diverse biological effects, adverse event profile, and the issues of tolerance, addiction, and safety. In particular, nAChRs have different effects under different conditions. Nicotine and partial agonists of nAChRs have the unique ability to regulate the network properties of groups of neurons and the immune system through the differential activation and desensitization of nAChRs on excitatory and inhibitory neuronal cell bodies and terminals . In inflWenjiZ contributed to writing—original draft preparation. HL contributed to writing—reviewing and editing. MZ contributed to investigation. QY contributed to data curation. ZH contributed to investigation. XP and WenjuZ contributed to project administration and supervision. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (grant number 81903319), Natural Science Foundation of Guangdong Province of China (grant number 2021A1515011220), Administration of Traditional Chinese Medicine of Guangdong Province of China (grant number 20211008), Special Fund for Young Core Scientists of Agriculture Science (grant number R2019YJ-QG001), Special Fund for Scientific Innovation Strategy—Construction of High-Level Academy of Agriculture Science (grant number R2018YJ-YB3002), Top Young Talents of Guangdong Hundreds of Millions of Projects of China (grant number 87316004), the foundation of director of Crops Research Institute, Guangdong Academy of Agricultural Sciences (grant number 202205) and Outstanding Young Scholar of Double Hundred Talents of Jinan University of China.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. 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Meiotic chromosomes endure rapid prophase movements that ease the formation of interhomologue recombination intermediates that drive synapsis, crossing over, and segregation process. To generate these fast moves, the meiotic telomere complex (MTC) enables telomere-inner nuclear membrane attachment during meiotic prophase I and transfers cytoskeletal signals via another complex: the LINC complex. Furthermore, disruption or mutations of any of the MTC genes  alters telomere association with the nuclear envelope leading to impairment of homologous pairing and synapsis, a meiotic arrest, and consequently to male infertility. To decipher the effect of TERB1, TERB2, and MAJIN missense mutations on protein structure, stability, and function, different bioinformatic tools were used in this study including VEP, Mutabind2, Haddock, Prodigy, Ligplot, ConSurf, DUET and MusiteDeep. In total, thirty mutations were predicted to be deleterious using VEP web server: seventeen for TERB1, eleven for TERB2, and two for MAJIN. All these single nucleotide polymorphisms were further analyzed and only 11 SNPs  were found to be the most damaging by at least six software tools and exert deleterious effect on the TERB1, TERB2, and MAJIN protein structures and likely functions. They revealed high conservation, less stability, and having a role in posttranslational modifications. This in silico approach provides information to gain further insights about variants that might affect stability, change binding affinity, and edit protein-protein interactions to facilitate their identification and functional characterization associated with male infertility. Telomeres have critical meiosis-specific functions in the reduction of chromosome numbers by assuring the integrity of the genome during meiosis. One such function is the attachment of telomeres to trans-nuclear envelope protein complexes that join telomeres to motor proteins in the cytoplasm. The active movement of telomeres and chromosomes during the first meiotic prophase is enabled by these trans-nuclear envelope linkages between telomeres and cytoplasmic motor proteins. Movements of chromosomes/telomeres facilitate the meiotic recombination process and ensure high fidelity pairing of homologous chromosomes which is a prerequisite for their correct segregation during the first meiotic division .Meiosis is a gametogenesis-specific cell division that entails unique chromosomal regulations such as pairing, synapsis, and homologous chromosome recombination. These processes are ensured by the dynamic chromosomal rearrangements that happen during meiotic prophase I which is prolonged and more complicated than mitosis. Indeed, it can last many days and account for up to 90% of the total time spent in meiosis . During The recently discovered tripartite complex comprising telomere repeats-binding bouquet formation proteins 1 and 2 (TERB1 and TERB2) and membrane-anchored junction protein (MAJIN) has been demonstrated to be involved in these processes by tethering telomere ends to the nuclear envelope (NE), transmitting cytoskeletal forces via the LINC complex to drive these rapid movements, and assembling of meiotic mammalian telomeres on the NE during prophase I .In mammals, the meiotic telomere complex (MTC) reuses and integrates the functions of two complexes, Shelterin and LINC, which otherwise play distinct significant roles outside of meiosis . The MTCMammalian telomeres contain a specific protein complex that accomplishes two functions: protecting the natural chromosome end from all factors of the DNA damage response within the cell and negative regulation of telomerase by segregation of its telomeric DNA substrate. Both functions of this complex refer to the name shelterin . The sheBecause of their TRF homology domains, the shelterin complex's subunits TRF1 and TRF2 perform a fundamental role by recognizing telomeric DNA, and by recruiting the other shelterin constituents onto telomeric DNA , 9, wherVia a perinuclear interaction, SUN1 (Sad1 and UNC84 domain containing 1) and KASH5 (Klarsicht/ANC-1/Syne/homology 5) proteins assemble into the mammalian meiosis specific LINC complex (linker of nucleoskeleton and cytoskeleton), providing the binding sites for telomeres on the inner surface of the nuclear envelope (NE) , 12 and Another protein complex consisting of TERB1 (telomere repeat binding bouquet formation protein 1), TERB2 (telomere repeat binding bouquet formation protein 2), and MAJIN (membrane anchored junction protein) has been demonstrated to build up a second actual linkage for telomere connection to the nuclear envelope by assuring meiotic telomere attachments in mammals , 22.The meiosis-specific telomere regulator TERB1 is a molecular scaffold that simultaneously interacts with SUN1 and meiotic cohesin subunit SA3 through its N-terminal ARM (armadillo) repeat domain and C-terminal Myb domain, respectively, providing telomere attachment to the inner nuclear membrane (INM) and driving the chromosome movement essential for homologous pairing and recombination , 22.Through a region surrounding its TERB2-binding site, TERB1 interacts directly with shelterin constituent TRF1, including a peptide interaction that resembles TIN2 binding to the TRF1 dimeric cleft , 23. ConTherefore, an interaction of the meiotic telomere complex with TRF1 seems to be transient in the cell. MAJIN–TERB2–TERB1 assembles on the nuclear envelope and endures TRF1-dependent recruitment of telomere ends during leptotene and zygotene , 22; CDKThus, in mice, knocking out the Terb1 gene disturbs the whole interaction network and affects homolog pairing, synapsis, and recombination, resulting in early spermatogenesis and oogenesis . HoweverMAJIN is a putative transmembrane (TM) protein, located at the inner surface of the NE; it serves as an anchoring component for the inner nuclear membrane through a transmembrane helix at its C-terminus . MAJIN hLately, computational analysis is designed to more accurately predict the impacts of mutations on proteins interactions. By using modern bioinformatics tools as a systematic in silico approach, our study is aimed at identifying the deleterious SNPs in the TERB1, TERB2, and MAJIN genes and predicting their significant pathogenic impact on the functions, stability, and structures of TERB1-TERB2 complex and TERB2-MAJIN complex.In this study, we carried out a systematic in silico approach using modern bioinformatics tools to predict deleterious SNPs in the TERB1, TERB2, and MAJIN genes and identify their significant pathogenic impact on the functions and structures of the TERB1, TERB2, and MAJIN proteins .https://www.ensembl.org): TERB1 (ENST00000433154.6), TERB2 (ENST00000340827.4), and MAJIN (ENST00000301896.6).The variant data related to the meiotic telomere complex components (TERB1-TERB2-MAJIN) was retrieved from Ensembl  was utilized. This tool provides an indication of the effect of the amino acid change using protein biophysical properties . These dThe Sorting Intolerant from Tolerant (SIFT) algorithm is predicting whether an amino acid substitution affects a protein function. It takes into account the position of the mutation as well as the type of amino acid change. SIFT assesses the probability that an amino acid at a particular location will be tolerated based on the most frequently tolerated amino acid. The substitution is predicted to be deleterious, if the normalized value is smaller than a cutoff. Scores range from 0 to 1. The smaller the score, the more likely the SNP has damaging effect –28.Polymorphism phenotyping (PolyPhen2) is a tool that predicts the possible impact of amino acid substitutions on the human protein structure and function using structural and comparative evolutionary considerations. PolyPhen2 results are available for human proteins. It classifies the substitution as probably damaging (score = 1 : 0) and possibly damaging or benign (score = 0 : 0) .Other pathogenicity predictor scores such as MutationTaster, MetaSVM, and MetaLR are available for human data via VEP plugins:MutationTaster is used for a quick assessment of the disease-causing potential of DNA sequence changes. Evolutionary conservation, splice-site changes, protein feature loss, and changes that could affect the amount of mRNA are all examined using MutationTaster, which predicts an alteration as one of the four possible types: “A” (disease causing automatic), “D” (disease-causing), “N” (polymorphism), or “P” (polymorphism automatic) , 31;MetaSVM (SVM (Support Vector Machine)) is a linear model that can be used to solve classification and regression problems. The theory behind SVM is simple: it finds and creates a line (hyperplane) that separates the data into different classes. Prediction using SVM approach in Ensembl database reveals “T” as tolerated or “D” as damaging or deleterious. The score cutoff between “D” and “T” is 0, and higher scores are more deleterious ;In MetaLR, to predict the deleteriousness of missense variants, logistic regression (LR) is used to combine nine independent variant deleteriousness scores with allele frequency information. Variants are categorized as “tolerated” or “damaging”; a score between 0 and 1 is also provided where variants with higher scores are more likely to be deleterious .https://www.rcsb.org/): The PDB ID for TERB1-TERB2 complex is 6J07 and for TERB2-MAJIN complex is 6GNY  by providing the following results: The ΔΔGbind  which is the predicted change in binding affinity induced by a mutation; in deleterious (yes or no), MutaBind2 server classifies a mutation as deleterious if ΔΔG ≥ 1.5 or ≤−1.5 kcal mol−1, ΔΔEvdw is the change of van der Waals interaction energy upon a mutation, ΔΔGsolv is the change of polar solvation energy upon mutation, and ΔΔGfold is the change of stability of protein complex upon mutation [In order to estimate the impacts of single mutations on protein-protein interactions (PPI), we performed computational analysis on the human crystal structure of TERB1-TERB2 and TERB2-MAJIN complexes using MutaBind2 . This tomutation .The chains of each complex (A and B of the 6J07 complex and C and D of the 6GNY complex) were separated using the UCSF CHIMERA program . Mutant The High Ambiguity Driven protein-protein Docking (HADDOCK) web server was employed to perform protein docking. The HADDOCK2.4 uses chemical shift perturbation data resulting from NMR titration experiments, mutagenesis data, and bioinformatics predictions . In the G) have greater binding affinity and correlates with mutations that stabilize the protein structures [Subsequently, the protein binding energy prediction (PRODIGY) web server was used to predict the binding affinity of the protein-protein complexes from their 3D structure , 40. ComLigplot software uses the 3D coordinates of a protein and its bound protein/ligand to automatically produce schematic diagrams. These diagrams show the pattern of interactions between the two molecules and are very useful for comparing different structures since they provide clear and helpful information of the intermolecular interactions and their strengths, including hydrogen bonds, hydrophobic interactions, and atom accessibilities .G). It further defines whether these changes increase or decrease the stability of the protein. Positive ΔΔG value denotes that protein stability increased, and negative ΔΔG means that protein stability decreased [The stability of the protein was checked using the Protein Stability Change Upon Mutation (DUET), which is an online server for an integrated computational approach to study missense mutations in proteins. It combines two complementary approaches (mCSM and SDM) in a consensus prediction, obtained by consolidating the results of the separate methods in an optimized predictor using Support Vector Machines (SVM) . DUET precreased .Site Directed Mutator (SDM) is an algorithm of statistical potential energy function that calculates a stability score based on environment-specific amino acid substitution frequencies within homologous protein families, which is comparable to the free energy difference between wild-type and mutant proteins .Mutation Cutoff Scanning Matrix (mCSM) relies on graph-based signatures used as a novel approach to the study of missense mutations and the prediction of their effects. These signatures encode distance patterns between atoms and are used to represent the protein residue environment and to train predictive models .To predict the evolutionary conservation of the amino acids in a protein sequence, we used a ConSurf bioinformatic tool that provides evolutionary profiles of each of the amino acids in the protein, based on phylogenetic relations between homologous sequences to reveal regions that are important for structure and/or function . The tooYASARA View is an open-source program for the molecular graphics, modeling, visualization, and analysis of the three-dimensional protein's structures. The structural deviations between the native and mutated models were analyzed using the YASARA View program, by measuring the root mean square deviation (RMSD) which is the average distance between the atoms of the superimposed proteins . Its valA posttranslational modification is crucial for cell signaling and affects the function of the protein. TERB1, TERB2, and MAJIN proteins' posttranslational modifications were predicted using MusiteDeep, an online service that includes a deep learning framework for predicting protein posttranslational modification (PTM) sites .The SNP dataset was collected from Ensembl database and had almost the same classification in the three studied proteins: about 96% involved germinal cells, while 3% concerned somatic cells. Different consequence types of these SNPs were detected: intron variant with the higher percentage , followed by missense variant , and other variants—3 prime UTR, 5 prime UTR, synonymous, splice region, and frameshift. In this study, seventy missense mutations that could have been mapped on the protein structure were included: 18 variants for TERB1, 31 variants for TERB2, and 21 variants for MAJIN .Thirty missense SNPs were predicted to be deleterious using SIFT (where tolerance index score was ranged from 0 to 0.02). The SNPs predicted by SIFT were validated by PolyPhen2, MutationTaster, MetaSVM, and MetaLR. In particular, PolyPhen2 results showed that eight AA substitutions  were predicted to be possibly damaging, while twenty-two SNPs were probably damaging: P649A, K632E, Y631S, Y631H, Y631N, H621Q, H621L, C618R, R605G, and S604G in TERB1 gene; W8R, G25R, G25V, F64S, G84S, G84D, P90S, G187W, D191N, and G198R in TERB2 gene; and R53H and R53C in MAJIN gene. The MutationTaster tool classified all these AA substitutions as deleterious .G ≥ 1.5: seventeen variants for TERB1, eleven variants for TERB2, and two variants for MAJIN  which correlates with lower binding affinity. These regions are represented by seven mutant complexes of TERB1-TERB2 complex  with a ΔΔG ≈ −10 kcal/mol and four complexes of the TERB2-MAJIN complex  with a ΔΔG ≈ −11.5 kcal/mol , Leu (2), Arg (8), Lys, and His) of TERB1 and amino acids , Lys (2), Thr, Asp (5), Gln, Ala (2), Asn (2), Glu (2), and Ile) of TERB2. In this complex, chain A (TERB2) revealed 12 hydrophobic interactions , Leu, Phe, Asn, His, Ile, and Glu) while chain B (TERB1) showed 14 hydrophobic interactions , Asn, Ala (2), Gly, Phe (2), and Tyr) (Ligplot result of the wild-type complex and Tyr) .R620C-W8R complex showed that hydrogen bonds formed by Gly, Val, Ser (3), Arg (8), Leu, His, Leu, Ile, Thr, Asp, Asn, and Gln amino acids were deleted, while new bonds with other residues (Asp (5), Asn, Thr (4), Gln, Arg, and His) were added. Further hydrophobic interactions have been added underlying the following Gly, Gln, Val, and Ser amino acids, whereas Ile, Cys, and Asn amino acids were eliminated.Mutated complex represented some differences in the hydrogen bonds and hydrophobic interactions: some contacts were added and others were removed. S604G-W8R; on the other hand, eight amino acids  were removed. Four hydrophobic contacts  were added, and eight were deleted .Nine amino acids  were added to hydrogen bonds of the mutated complex S604G-G25V complex showed that hydrogen bonds constituted by Gly, Asp, Asn, Arg, Tyr, Gln, Thr, and His residues were added and Leu, Val, Asp, Glu, Thr, Asn, Arg, Gln, His, Lys, Ser, and Ile were removed. Some hydrophobic interactions involving Gln, Lys, Ser, Arg, Val, and Pro have been added, and Arg, Cys, Asn, Val, Lys, Glu, Pro, and His were eliminated.C618R-F64S revealed that hydrogen bonds formed by Asp, Ile, Asn, Thr, Gly, Gln, Arg, and His amino acids were added, while those formed by Gly, Val, Ser, Arg, Lys, His, Leu, Ile, Ala, Thr, Glu, Asp, and Gln were canceled. For hydrophobic contacts, Lys, Gln, Pro, Val, and Arg amino acids were added and Arg, Cys, Asp, His, Pro, Asn, and Gly amino acids were deleted.The mutated complex C618R-G84D. Nine hydrophobic interactions  were added, while four  were removed.Eight amino acids  were added, and nine  were removed in the hydrogen bonds of the mutant complex Y631N-G84D revealed that hydrogen bonds formed by Asp, Tyr, Asn, Arg, Cys, Ile, His, and Phe amino acids were added and hydrogen bonds formed by Leu, Val, Asp, Glu, Thr, Gly, Ser, Lys, Arg, and Ile amino acids were removed. For hydrophobic contacts, six amino acids  were added while nine amino acids  were canceled  were added, and five  were removed.TERB2-MAJIN undergoes 20 hydrogen bonds formed between amino acids , Arg (6), Phe, His (3), Asn, Ala (2), and Gly) of MAJIN chain and amino acids (Leu (3), Asp (3), Phe (3), His (2), Tyr (3), Gly (3), Lys, and Ile (2)) of TERB2 chain. MAJIN protein carries 19 hydrophobic contacts (Leu (5), Ser (3), Phe (3), Ile, Ala, Thr, Val, Asn, Gln, Asp, and Gly) while 13 hydrophobic interactions (Gly (2), Ala, Val, Ile, Glu, Tyr, Met, Lys (2), Leu (2), and Ser) were detected in TERB2 protein.The second wild complex WT TERB2-R53H showed that six amino acids  were added to hydrogen bond interactions, while three  were removed. The hydrophobic interactions listed two amino acids (Ile and Asp) as deleted and only one amino acid (Pro) as added.The mutated complex MAJIN-L189P, and five were removed . Hydrophobic contacts added three new residues  to the list, while four residues Leu, Ile, Val, and Gly were taken off . Some hydrophobic interactions implying Gly, His, and Asn have been added, and Asp, Val, and Ile have been eliminated , and six were canceled . In the hydrogen bonds, six amino acids  were added, while one (Gln) was removed.Five amino acids  were added to hydrogen bonding of the mutated complex aken off . The mutiminated . About tDUET, ConSurf, and MusiteDeep web servers were used to identify, respectively, the mutation effects on protein stability and the conserved domains in proteins and predict directly the posttranslational modification (PTM) site from the raw protein sequence. The mutated structures were also analyzed by YASARA View, with regard to evaluate conformational variations by calculating the RMSD.The results of mCSM, SDM, and DUET algorithms showed that 23 amino acids substitutions were recorded as destabilizing by the three algorithms: P649A, K632E, Y631S, Y631H, Y631N, I624T, H621Q, C618R, F607V, R605Q, R605G, C592Y, and C592R of TERB1; W8R, F64S, G84S, G84D, P90S, G25R, G187W, L189P, and G198R of TERB2; and R53C of MAJIN . FurtherCompared to those in nonconserved regions, amino acids located in conserved regions were predicted to be highly damaging. ConSurf predicts amino acids to play structural or functional roles based on conservation and solvent accessibility. Residues are predicted as functional when they are highly conserved and exposed and as structural when they are highly conserved and buried.Phylogenetic conservation analysis of TERB2 protein showed that G25, P90, G187, D191, and G198 are highly conserved residues and predicted to be exposed and functional. On the other hand, W8, F64, G84, and L189 are highly conserved residues and predicted to be buried and structural. Finally, R53 is highly conserved and predicted to be exposed and a functional residue in MAJIN protein .Analysis of TERB1 protein from the ConSurf server revealed that P649 and S604 are highly conserved residues and predicted to be exposed and functional, while I624, H621, C618, F607, and C592 are highly conserved residues and predicted to be buried and structural. K632, R620, S608, and R605 are exposed residues: R620 is variable, while K632 and S608 are conserved. Y631 is a conserved and buried residue .In our study, 29 mutations out of 30 showed higher RMSD values which indicate greater variation between wild and mutant protein structures. Therefore, four variants C592R, G25V, G198R, and R53H reflected maximal structural dissimilarity with TERB1-TERB2 complex and TERB2-MAJIN complex  and were therefore used for superimposing . One resMusiteDeep is applied to predict the effect of SNPs on posttranslational modification (PTM) process of the human TERB1, TERB2, and MAJIN proteins. MusiteDeep identified sites for O-linked glycosylation: R605 in TERB1 protein and W8, G84, G187, L189, D191, and G198 in TERB2 protein sequence . S-PalmiA cumulative score (CS) was calculated for the 30 damaging variants based on seven in silico tools used for predicting the effect of SNPs  to further understand and justify how the most deleterious mutations were identified in this study .All of the listed 30 SNPs were predicted as deleterious or damaging by SIFT (SIFT < 0.05), PolyPhen2 (possibly and probably damaging SNPs), and Mutabind2 (ΔGG ≥ 1.5) ; and onlThe results of several recently published papers have contributed to our understanding of the meiotic telomere complex assembly in mammals and its possible interaction with Shelterin and LINC complexes, thus providing a significant advance in the identification of the importance of meiotic telomeres complex which has been gradually elucidated and clarified by various authors. In 2014, Shibuya et al. showed that TERB1 = CCDC79 structure contains two armadillo repeats at the N terminus, a coiled coil motif in the middle region and, remarkably, a Myb domain at the C terminus, a structure which resembles that of TRF1. At this point, many roles were dedicated to TERB1 . It is iIn addition to TERB1, two downstream meiotic proteins, TERB2 and MAJIN, are recruited to the meiotic telomeres by TERB1 in mammalian meiosis . ShibuyaIn 2019, Wang et al. concluded that the intact MTC complex is required for gametogenesis and fertility, and targeted disruption of the complex induces disordered synapsis and impairs meiotic double-strand break (DSB) repair and abolishes telomere-NE linkage, together leading to a complete meiotic arrest in prophase I in both sexes. Hence, both the MTC complex and SUN1 (the LINC complex) contribute to the stable telomere-NE association and are important for efficient progression of prophase I .Last year, Salas-Huetos et al. strongly confirmed that disruption of any of the three genes coding for the meiotic telomere complex (MTC) can cause NOA in men. It has been demonstrated previously that disruption of Terb1 in the mouse results in meiotic arrest and impairment of homologous pairing and synapsis, ultimately resulting in infertility in both males and females . LikewisTaken together, they showed that the disruption or mutations of any of the MTC genes impairs telomere association with the inner nuclear membrane, which is requisite for chromosomal synapsis and chromosome movement during meiosis. Failure of these events triggers meiotic checkpoints leading to a meiotic arrest and consequently to male infertility/NOA . The maiBioinformatic analysis provides powerful insights into understanding and predicting the effects of mutations on protein structures, including how mutations alter protein stability, binding energy, binding affinity, evolutionary conservation and interactions with other proteins and their relation to susceptibility of genetic diseases. In the current study, various algorithms, namely, VEP tools , Mutabind2, Haddock, Prodigy, Ligplot, DUET, ConSurf, and MusiteDeep, were used to identify the most deleterious SNPs of the TERB1, TERB2, and MAJIN genes. Accordingly, eleven SNPs out of thirty  scored the highest cumulative scores (CS = 6) predicted by different bioinformatic tools and were located in conserved regions with a score conservation of 9, which may affect the structure and function of proteins .Protein-protein docking aims to anticipate molecular complex's structure given the individual solved structures of its constituents, which can be determined experimentally or predicted. In the present study, docking was performed by HADDOCK web server that displays results as cluster's structures, where the first top cluster produced is deemed reliable and should be downloaded in order to be loaded on the PRODIGY web server to calculate binding energy. Binding affinity refers to the strength or interaction energy of the binding between the protein and its partner. According to the results of heatmap, the lighter colored areas represent the complexes with higher binding energy and less binding affinity: R620C-W8R, S604G-W8R, S604G-G25V, C618R-F64S, C618R-G84D, Y631N-G84D, and WT TERB1-G25R of TERB1-TERB2 complex and WT TERB2-R53C, WT MAJIN-L189P, WT MAJIN-D191N, and L198P-R53C) of TERB2-MAJIN complex . GeneralMissense mutations can affect protein-protein interactions via various molecular mechanisms, including changing folding free energy of interacting partners, modifying protein stability, or disrupting noncovalent interactions essential for complex formation and function. Binding affinity is also influenced by noncovalent interactions between biomolecules, such as the loss or gain of hydrogen bonding and/or hydrophobic contacts in the mutated complex compared to the wild-type complex. After visualization by Ligplot, we concluded that the eleven previous complexes with the lower binding affinity showed differences in protein-protein interactions where some contacts were added and others were deleted, which explains the diminution of binding affinity within these complexes .G) predicted by the 3 algorithms which indicate that they destabilized the protein's stability, except for the S604G that was destabilizing only by two algorithms  as they were agreed by six software. Combinations of multiple in silico tools provided information required to predict the effects of these mutations on the functional and structural deviations of TERB1, TERB2, and MAJIN proteins. This work has highlighted the need to take into account the type of protein-protein interactions when characterizing the variants within the complexes. Hence, our result added to previous knowledge and supported earlier findings that may be helpful for further understanding the role of meiotic telomere complex (MTC) in male infertility. Nevertheless, bioinformatics tools cannot replace conclusive experiments, and their results should be verified by supplementary data.


WFS1 and CISD2.Wolfram syndrome (WFS) is characterized by deafness, diabetes mellitus, and diabetes insipidus along with optic atrophy. WFS has an autosomal recessive mode of inheritance and is due to variants in We evaluated the underlying molecular etiology of three affected members of a consanguineous family with hearing impairment, bicuspid aortic valve, diabetes mellitus and insipidus, clinodactyly, and gastrointestinal tract abnormalities via exome sequencing approach. We correlated clinical and imaging data with the genetic findings and their associated phenotypes.CDK13, a gene previously associated with autosomal dominant congenital heart defects, dysmorphic facial features, clinodactyly, gastrointestinal tract abnormalities, intellectual developmental disorder, and seizures with variable phenotypic features.We identified a homozygous missense variant p.(Asn1097Lys) in CDK13 and suggest that this gene causes an autosomal recessive disorder with hearing impairment, bicuspid aortic valve, diabetes mellitus and insipidus, clinodactyly, and gastrointestinal tract abnormalities.We report a homozygous variant in  WFS1, for Wolfram syndrome type 1, and CISD2, for Wolfram syndrome type 2, are inherited with an autosomal recessive (AR) mode of inheritance and encode for wolframin and CDGSH iron-sulfur domain 2 proteins, respectively, and have high expression in brain, lung, heart, and pancreas , which lies within the region of homozygosity. Sanger sequencing of DNA samples from the remaining family members demonstrated that the variant segregated with the syndrome and had a LOD score of 3.11.We report on a consanguineous Pakistani family with an AR WFS-like phenotype that segregates a CDK13, expressed ubiquitously in human tissues, is part of the cyclin-dependent kinase family that regulates gene transcription, alternative splicing of RNA, and C-terminal domain (CTD) phosphorylation, along with CDK12 and cyclin K . It is located just C-terminally of the protein kinase domain  variant in Cdk13 is expressed in cochlea and utricle cells over four developmental stages: E16, P0, P4, and P7. In cells surrounding the cochlea and utricle, highest levels of expression were seen at P0. In hair cells, highest expression levels were seen at stage P0 in cochlea and at P4 in utricle  is displayed in Fig. The crystal structure of  PDB ID: EFQ is diCDK13 that segregates with a Wolfram-like syndrome in a consanguineous Pakistani family. The three affected family members present with severe to profound bilateral SNHI, insulin-dependent diabetes mellitus and insipidus, bicuspid aortic valve, clinodactyly, and GI tract abnormalities. One affected member also presented with seizures due to epilepsy. Although Wolfram syndrome was suspected, all affected individuals also had bicuspid aortic valves with no other structural and functional heart defects as well as clinodactyly which are inconsistent with WFS-1 and −2. All three affected siblings are within the age of onset of optic atrophy. Therefore, it cannot be ruled out that the two living affected family members will develop optic atrophy as they grow older, although typically WFS patients have onset <15 years of age, while for AD Wolfram-like syndrome onset of optic atrophy has been reported to occur as late as 20 years of age [We performed whole-exome sequencing and identified a homozygous missense variant in s of age .Overlapping features observed between family 4743 and CHDFIDD include cardiac septal defects, clinodactyly, and GI tract abnormalities that were observed in the three affected siblings . SeizureCDK13 is ubiquitously expressed in human tissues with highest levels of expression in the cerebellum, thyroid gland, colon, skin, ovary, and testis [CDK13 expression [Cdk13 is strongly expressed in the retina, testes, ovary, uterus, gallbladder, heart, thyroid gland, and kidney [Cdk13 is prominently expressed in mice inner ear from developmental stage E16 to P7, with varying levels of expression observed in the spiral and vestibular ganglion from stages E12 to P15 as well.d testis , 44. It d testis . Fetal bpression . In miced kidney . In siliCDK13, implicated in CHDFIDD with varying phenotypes, are clustered in the protein kinase domain that binds ATP and magnesium [CDK13 heterozygous pathogenic, likely pathogenic or variants of unknown significance (VUS), mostly loss of function (LoF) variants  with wide phenotypic heterogeneity have been evaluated [N = 4), p.(Asn842Ser) (N = 21), p.(Ala162fs) (N = 3), and c.2601–2 A>G (N = 2) [CDK13 variant to be associated with an AR phenotype. Most AD CDK13 variants have been predicted to lead to a LoF, although functional studies have not been performed to validate the classification. So far, of the 42 known cases presenting AD CHDFIDD due to CDK13 variants one patient has only mild or no ID that was confirmed by Wechsler Nonverbal (WNV-IQ) test [De novo pathogenic mutations in agnesium , 42, 46.valuated . Notablyvaluated . The maj (N = 2) , 49, wittm1a/tm1a knockout mice after stage E15.5 due to chronic heart failure, suggesting their role in transcriptional regulation during embryonic development [tm1a/tm1a knockout mice embryos along with underdeveloped brain, lung, liver, and kidney [tm1a/tm1a knockout mice between E14.5 and E16.5 was severely affected leading to kidney failure, while liver function remained intact [tm1a/+ mice were normal and healthy. In a study of Cdk13tm1b(EUCOMM)Hmgu mice, preweaning lethality with complete penetrance was observed for homozygous Cdk13tm1b(EUCOMM)Hmgu mice, while heterozygous Cdk13tm1b(EUCOMM)Hmgu mice displayed heightened startle reflex, abnormal lens morphology, and cataracts [Embryonic lethality was observed in homozygous Cdk13elopment . Heart dd kidney . Gross mataracts .CDK13 segregating in a Pakistani family with severe to profound SNHI, bicuspid aortic valve, diabetes mellitus and insipidus, clinodactyly, and GI tract abnormalities which suggests that besides CHDFIDD, CDK13 also likely causes an AR Wolfram-like syndrome which includes cardiac defects, clinodactyly, and GI tract abnormalities. The identification of additional families with AR Wolfram-like syndrome due to CDK13 will aid in determining the complete disease spectrum and also serve to confirm the role of CDK13 in the etiology of AR Wolfram-like syndrome. The identified variant was submitted to ClinVar (Accession Number: SCV001468320) as a VUS.In conclusion, this study has identified a novel homozygous missense variant, c.3291 A>C: p.(Asn1097Lys), in Supplementary Acknowledgements and FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3