Seven probands carry causative variants in well-known genes associated with CA or cerebellar hypoplasia: SETX, CACNA1G, CACNA1A, CLN6, CPLANE1, and TBCD. The remaining three cases deserve special attention; they harbour variants in MAST1, PI4KA and CLK2 genes. MAST1 is responsible for an ultrarare condition characterised by global developmental delay and cognitive decline; our index case added ataxia to the list of concomitant associated symptoms. PIK4A is mainly related to hypomyelinating leukodystrophy; our proband presented with pure spastic paraplegia and normal intellectual capacity. Finally, in a patient who suffers from mild ataxia with oculomotor apraxia, the de novo novel CLK2 c.1120T>C variant was found. The protein expression of the mutated protein was reduced, which may indicate instability that would affect its kinase activity.Cerebellar atrophy (CA) is a frequent neuroimaging finding in paediatric neurology, usually associated with cerebellar ataxia. The list of genes involved in hereditary forms of CA is continuously growing and reveals its genetic complexity. We investigated ten cases with early-onset cerebellar involvement with and without ataxia by exome sequencing or by a targeted panel with 363 genes involved in ataxia or spastic paraplegia. Novel variants were investigated by  The classic role of the cerebellum is to be an efficient modulator of movements. Cerebellar dysfunction is traditionally related to cerebellar motor syndrome, which is characterised by impairments of gait, limb coordination, speech, and ocular movement control . Along wde novo disease-causing mutations transmitted in an autosomal dominant (AD) manner is appreciated [https://hpo.jax.org/app; accessed on 14 September 2023), yields associations with 579 genes.Early-onset ataxias usually present with a relevant disability, including imbalance, poor coordination, developmental delay, and intellectual disability (ID) ,4. CerebEXOSC3, pontocerebellar hypoplasia type 1B) [Among the cerebellar ataxias, we find ARCAs  or SCARs (AR Spinocerebellar Ataxias), and ADCAs  or SCAs (AD Spinocerebellar Ataxias), in which ataxia is a prominent clinical feature or is a sign included in the clinical phenotype ,13,14. Ttype 1B) . The lisSETX, CACNA1G, CACNA1A, CLN6, CPLANE1, and TBCD, with well-known-disorders, new clinical phenotypes related to not-so-common genes, MAST1 and PI4KA, and a novel candidate gene, CLK2, which may act as causative gene or contribute to the clinical phenotype. Additionally, in silico or experimental studies were performed to ascertain the pathogenicity of novel variants, which has been demonstrated essential to achieve conclusive results [We describe here ten probands who show CA with or without ataxia, in which a brain magnetic resonance imaging (MRI) was performed due to a motor impairment or early signs of a neurological disease. These patients were selected from a national cohort of children with clinical signs of an early-onset movement disorder and/or ataxia, and CA identified by brain MRI. Causative genes are involved in ataxia phenotypes but are also associated with channelopathies, neuronal ceroid lipofuscinosis, Joubert syndrome (JS) and encephalopathies, which highlights the wide spectrum of genotypes associated with CA. In this cohort, we found relatively frequent genes,  results ,16,17,18SETX, SETX, which has been widely associated with SCAR previously , there is no experimental structure, and the model generated by AlphaFold displays a predominant absence of secondary structure. However, the folding for the C-terminal region of senataxin, where the missense clinical mutation p.I1942T is located, it has been predicted to contain a helicase domain with homology to human RENT1, the immunoglobulin μ-binding protein 2 (IGHMBP2), and yeast-splicing endonuclease 1 (SEN1) [The patient MD-359 SETX, , was an eviously . The varCACNA1G, CACNA1G c.2881G>A (p.A961T) variant was observed in the patient in a heterozygous state; his parents did not carry the variant. CACNA1G c.2881G>A has been previously reported  change was identified in the patient. Further segregation analysis showed that this mutation was de novo.The patient MD-309 CACNA1A,  was a 12CACNA1A, de novo deletion of c.234_235delCT (p.F79Pfs*22) in CACNA1A, was, to date, not associated with disease in heterozygosis in the patient, predicted as pathological according to the ACMG /AMP (Association for Molecular Pathology) criteria [The patient MD-556 CACNA1A, , a 14 yecriteria   in heterozygosis, predicted as damaging according to the ACMG/AMP criteria  compared with the rest of the samples suggesting a heterozygous intragenic deletion in the proband MD-392. By PCR and subsequent Sanger sequencing, we established that the deletion was of 45,305 bp  from intron 15 to intron 32. Further transcript analysis showed the absence of exons 16 to 32, suggesting a change in the reading frame and a premature termination codon  variant was detected by WES-proband in homozygosis. Her healthy mother carried the TBCD c.3099C>G in heterozygosis and her paternal uncle did not (sample from the MD-297′s father was not available). TBCD c.3099C>G was previously associated with early-onset progressive encephalopathy  proteins of the spliceosomal complex. The proband has mutated the residue Y374. To test whether the detected p.Y374H affects the CLK2 protein levels suggesting instability of the mutated protein, as previously described for the variants p.K192R and p.T343A in mice [Finally, the patient MD-436 CLK2, , was fir in mice , we tranThe number of disorders that present with CA is ever-increasing with an extremely broad clinical spectrum, from non-progressive developmental disorders to neurodegenerative conditions ,9,10,11.SETX, CACNA1G, CACNA1A, MAST1, TBCD, CLN6, and PI4KA, highlighting the heterogeneity of associated genotypes with a clear progression of CA over time. Additionally, in three patients a white matter involvement was associated with CA carrying mutations in CLN6, MAST1 and PI4KA genes. A suggestive MRI pattern was identified in two patients with mutations in MAST1, associated with pseudofusion of the putamen and caudate, and mega-corpus callosum size, and in TBCD, causing hypomyelination, and CA and callosal thinning, which are hallmarks of this progressive disorder.In this cohort, CA, as determined in serial neuroimaging, is present in eight individuals with variants in 7 genes: SETX are annotated in HGMD® , some of them especially recurrent, as the mutation identified in homozygosis in the girl MD-359, c.5825T>C. Modelling of the protein showed that the mutation may destabilize the folding affecting its stability. Mutations in SETX transmitted in an AD manner are involved in amyotrophic lateral sclerosis-4 (ALS4), and with an AR mode of inheritance, are responsible for SCA with axonal neuropathy (SCAN2), which is characterised by progressive CA, along with sensorimotor neuropathy, occasional oculocephalic dissociation and/or oculomotor apraxia, strabismus, chorea, dystonia, and elevated serum α-fetoprotein levels [SETX-associated ataxia and CA in 96% [SETX may cause neuronal degeneration through dysfunction of helicase activity or other steps in RNA processing [SETX. So far, 338 different variants in n levels ,29. Polyocessing ,31. MD-3CACNA1G leads to SCA42, a slowly progressive ataxia, characterised by severe motor, cognitive impairment, and CA, with variable features such as facial dysmorphisms, digital anomalies, microcephaly, and epilepsy [de novo CACNA1G c.2881 G>A change in heterozygosis, a well-known mutation associated with SCA42. Modelling of the mutated protein showed that the change p.A961T may affect the intracellular segment required for binding to the β regulatory subunit of this channel, which may impair the proper functioning of the channel. MD-353 is at present, a 17 year-old boy, with a stable ataxia course and no epilepsy, who at age of 13 months displayed cerebellar reduced volume that was associated with CA afterwards. He showed moderate cognitive impairment and attention deficit. Strikingly, the proband suffered from congenital arthrogryposis, which is not a common feature related to ataxias [VRK1 mutations that occasionally includes ataxia [Implication of ion channels in genetic ataxias associated with CA is long known, given the importance of ion balance, notably that of calcium ions, in cerebellar pathology. In fact, in mutational screening of clinical series of patients with cerebellar ataxia, the frequency of channelopathies is relatively high ,33. Thisepilepsy ,34. CACNepilepsy ,34. MD-3s ataxia . CACNA1A. Episodic ataxia type 2 (EA2), developmental and epileptic encephalopathy 42 (DEE42), and familial hemiplegic migraine are also due to CACNA1A variants. EA2 or SCA6 can be caused by abnormal (CAG)n expansions as well as point mutations. The CACNA1A-associated phenotypic spectrum is really much more wider comprising early developmental delay, autism spectrum disorders, epileptic encephalopathy, and early onset paroxysmal dystonia [CACNA1A are known . The CACNA1A gene encodes the transmembrane pore-forming subunit of the P/Q-type or CaV2.1 voltage-gated calcium channel, which functions as a transcription factor that mediated cerebellar development [CACNA1A probands carry c.3042C>G (MD-309) or c.234_235delCT (MD-556), in heterozygosis, which have not been previously associated with disease. Another more frequent ataxia due to defects in a calcium channel is SCA6, caused by mutations in dystonia , as reveelopment . The CACCLN6 lead to NCL type 6A and type 6B. CLN6 encodes for a transmembrane protein that is expressed in in adult and embryonic brain and in peripheral tissues of mouse and human [CLN6-associated phenotype fits with this clinical picture and fatal outcome with premature death. Ataxia is a common additional neurological sign, and even may be the primary clinical feature at symptoms onset, which may mislead the diagnosis of NCL [CLN6-associated phenotype, including CA and abnormal cerebral white matter morphology, which are also observed in CLN6 patients [® Professional 2023.1; accessed on 14 September 2023). Neuronal ceroid lipofuscinosis (NCLs) are a group of inherited lysosomal storage disorders with a notable genetic heterogeneity (at least 15 genes involved) and a complex phenotype. Deleterious mutations in nd human . CLN6 dynd human . The clind human . The CLNs of NCL . Other fs of NCL . Patientpatients . Both stMAST1 alterations lead to a mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations. The clinical picture comprises non-verbal moderate to profound ID, and hypotonia in all of them [MAST1 . McMichael et al. [de novo novel missense mutation in a patient with diplegic cerebral palsy, but with no further clinical information. Tripathy et al. [MAST1 mutations have been reported with atypical presentations, such as absence of cerebellar hypoplasia, predominant bilateral polymicrogyria, dysmorphic features, short stature, epileptic encephalopathy, and hypogonadotropic hypogonadism [de novo novel change predicted as damaging according to the ACMG/AMP criteria [MAST1 mutations. He is currently a nonverbal teen with a severe ID, hypotonia, and truncal ataxia, but a cortical development malformation is lacking in neuroimaging. Additionally, it was disclosed a bilateral thinning of the anterior arm of the internal capsule resembling a pseudo-fusion of putamen and caudate nuclei, not previously reported. With MD-548, we describe the novel MAST1 c.1672G>C mutation, expanding the clinical picture of this ultrarare neurodevelopmental disorder, suggesting that ataxia may be more frequently present but masked as persistent hypotonia in other cases.  of them . To our l et al.  reportedy et al.  delineatgonadism ,48,49,50CPLANE1. Both mutations investigated by transcript analysis revealed that altered the proper splicing causing the loss of exons. Damaging variants in CPLANE1 can cause JS or more frequently, orofaciodigital syndrome VI (OFD6). Around 35 genes are involved in JS and related disorders, which are characterised by a pathognomonic cerebellar and brainstem malformation on brain MRI, the so-called molar tooth sign. JS is a multisystem syndrome with a complex phenotype that affect different body systems , clinically characterised by hypotonia, ataxia, developmental delay or ID, oculomotor apraxia, and apnea or hyperapnea [CPLANE1 (ciliogenesis and planar polarity effector) protein is required for cilium assembly in ciliated cells [The proband MD-392 was a compound heterozygous for c.7588+7A>G and c.2747-1981_6172-78del in perapnea . MD-392 ed cells . Abnormaed cells ,54. Uponed cells .Early-onset progressive encephalopathy with brain atrophy and a thin corpus callosum (PEBAT) is caused by damaging variants transmitted in an AR fashion in TBCD. TBCD is one of the five tubulin-specific chaperones (TBCA-E) needed for the reversible assembly of the α-/β-tubulin heterodimers, which are involved in crucial cell functions such as division or intracellular transport . PatientPI4KA cause a variable phenotypical spectrum ranging from severe neurodevelopmental disorder with spasticity, hypomyelinating leukodystrophy, and brain abnormalities (NEDSPL) to pure spastic paraplegia type 84 (SPG84) [PI4KA and the transcript analysis performed for c.3845C>G revealed that may affect the proper splicing. Two patients have been previously described with a similar clinical picture without cognitive or language impairment, but both did not show supratentorial white matter hypomyelination as ours, so a longer follow-up must be needed to ascertain the milder phenotype [AR mutations in  (SPG84) . Additio (SPG84) . MD-610 henotype . de novo novel variant CLK2 c.1120T>C (p.Y374H) in heterozygosis. To date, no Mendelian disorders have been associated with CLK2 mutations. A translocation t , affecting CLK2 and PAFAH1B3, was reported in a female with ID, ataxia and brain atrophy [PAFAH1B3, which resulted to be inactive and hence, without capacity of interacting with LIS1 , a member of the heterotrimeric G protein complex PAF-AH1B. CLKs belong to the dual-specificity protein kinase family and play an essential role in the regulation of the transcript splicing via the phosphorylation of the serine/arginine-rich (SR) proteins and subsequently modulates the alternative splicing of precursor mRNA (pre-mRNA) [The proband MD-436 suffered from a mild ataxia, oculomotor apraxia, borderline IQ, and language delay, with global improvement over time. A single brain MRI revealed a reduced vermian volume with enlarged interfolia spaces at two years of age. In the patient MD-436 investigated by WES-trio, the only variant of interest was the  atrophy . The molre-mRNA) . The patre-mRNA) . The CLKre-mRNA) . We havere-mRNA) ,63. Recere-mRNA) ,63, whicTen probands were selected based on the presence of CA associated to ataxia, ID and/or additional neurological signs and symptoms. Phenotyping included standardised assessments of ataxia, movement disorders, and other neurological symptoms such as spasticity, muscle weakness or signs of peripheral neuropathy. An intelligent quotient was obtained by WISC-IV . MRI stuEight out of ten patients went for WES-proband or WES-trio, depending on the samples sequenced) based on a xGen Exome Research Panel with custom-designed capture probes  at Blueprint Genetics  or KAPA HyperExome  at CNAG . MD-309 and MD-392 were investigated using a custom panel for ataxia and spastic paraplegia (Panel SPGAtaxia-363) based on SureSelect Custom Panel  including 363 genes (gene list upon request). Bioinformatics analyses  were carried out in-home as previously described . To clasSanger sequencing on an ABI Prism 3130XL analyser  was performed for validation by PCR and segregation analysis of the genetic candidate variants identified in the bioinformatics analysis. Primers are available upon request.Copy number variants (CNVs) were investigated using the coverage-based DECoN bioinformatics tool. BAM files from samples with consistent exon CNVs calls were visually inspected in the Integrative Genomics Viewer (IGV) to find additional support for the calls and putative breakpoints, that were further determined by PCR and Sanger sequencing of DNA samples. https://aphafold.com; accession code Q9BTW9) [https://pymol.org/2/).The structural analysis of SETX was based on the experimental structure of the yeast SEN1 helicase domain (PDB entry 5mzn) , as well Q9BTW9) ,67. For  Q9BTW9) , where sCPLANE1, c.7588+7A>G and c.2747-1981_6172-78del, as well as the variant c.3845C>T identified in PI4KA : 1/1000 and anti-GAPDH (0411) : 1/8000. The CLK2 cDNA (NM_001294338.2) cloned in the pcDNA3.1+/C-(K)-DYK vector (oHu28699D), tagged with DYK in C-ter, was obtained from GenScript . The CLK2 p.Y374H mutation was generated with specific primers  using thNew genes and new variants are constantly identified thanks to the increased availability of exome and genome sequencing together with new methods such as long-read sequencing. To determine if a genetic variant is deleterious or not is essential. Moreover, most conditions are rare disorders characterised by genetic heterogeneity and allelic diseases. Definitely, diagnosis is a challenge. Therefore, the characterisation of new cases that support the known phenotypes, reveal new clinical pictures, and/or show new diseases due to genes not yet associated with Mendelian diseases is crucial to improve the genetic diagnosis, mainly in childhood. In this work, we contributed to the landscape of early-onset cerebellar atrophy with ten new cases and nine different genes, including additional experiments to gain evidence of the pathogenicity of several variants.


Objectives: The impact of non-steroidal anti-inflammatory drugs (NSAIDs), conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) and tumor necrosis factor inhibitors (TNFi) on the outcomes of mild-moderate COVID-19 in patients with ankylosing spondylitis (AS) remains unclear. This study aimed to evaluate the effects of NSAIDs, csDMARDs, and TNFi on AS patients with mild-moderate COVID-19.Methods: This cohort study utilized patient-reported PCR/antigen tests to determine the occurrence of COVID-19 and assessed clinical manifestations to determine its severity. The study focused on two primary outcomes: an increased number of COVID-19 symptoms and a prolonged disease course (longer than 10 or 28 days). Modified Poisson regression was performed to analyze the association between exposures and outcomes.Results: A total of 521 patients were included in the analysis. The median age was 34.8 (inter-quartile range: 27.2–46.7), with 420 (80.6%) being men. Among the patients, 52 (10.0%) had comorbidities and 443 (85%) had been vaccinated. After adjusting for confounding factors, there was no significant association between csDMARDs or TNFi and the presence of more than 5 symptoms in mild-moderate COVID-19 (adjusted relative risk (RRa) 1.08, 95% CI: 0.84–1.40 or 1.09, 0.92–1.29 for csDMARDs or TNFi, respectively), whereas the prevalence of experiencing more than 5 symptoms increased in patients with NSAID monotherapy . Similarly, there was no significant association with having more than 10 symptoms . Patients who had pre-existing use of NSAIDs, csDMARDs and TNFi had similar odds of experiencing a disease course longer than 10 days  and longer than 28 days  compared to those not using medication.Conclusion: AS patients treated with csDMARDs or TNFi did not show inferior outcomes in terms of symptom burden or recovery compared to those not using medication in mild-moderate COVID-19. The observed inverse association between pre-existing NSAIDs use and COVID-19 symptom burden in AS deserves further investigation. Despite the World Health Organization (WHO) declaring an end to the COVID-19 pandemic as a public health emergency , it had Tumor necrosis factor inhibitor (TNFi) is a widely used bDMARD in the treatment of AS and is known for its immunosuppressive properties. Traditionally, TNFi has been associated with an increased risk of infection . HoweverThis retrospective cohort study was conducted at the outpatient rheumatology clinics of the First Medical Center of the Chinese People’s Liberation Army (PLA) General Hospital, a tertiary referral center in Beijing, China. Patients attending the clinics were invited to participate in the study and complete questionnaires that included demographic data, AS disease characteristics, and COVID-19 infection details. Besides, evaluation of AS disease activity  , functioCOVID-19 infection details included the results of SARS-CoV-2 PCR or antigen tests, vaccination status, COVID-19 symptoms, and the time taken for patients to recover. Patients aged 18 years or older were enrolled in the study from 20 December, 2022, to 31 March, 2023, if they met the 1984 modified New York criteria for AS  and had This study was conducted in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines and complied with the Declaration of Helsinki.NSAIDs, TNFi, and csDMARDs exposure was defined as patients being prescribed NSAIDs, TNFi, and csDMARDs within 12 months before the index date, respectively. TNFi included etanercept and its biosimilars, adalimumab, and infliximab. The range of csDMARDs in this study included sulfasalazine, methotrexate, leflunomide, thalidomide, and iguratimod. Given the clinical practice and the real-world background of the study, patients taking AS medications were grouped as NSAIDs monotherapy, NSAIDs + csDMARDs, csDMARDs monotherapy, and TNFi (with or without NSAIDs/csDMARDs).Covariates such as age, sex, comorbidity, body mass index (BMI), smoking, alcohol consumption, and vaccination status might be associated with different outcomes in COVID-19. Therefore, they were considered confounding factors and adjusted for in further multivariable analysis. Comorbidities included diabetes, cardiovascular disease , and chronic obstructive lung disease (COPD). Vaccination status was classified as unvaccinated, partially vaccinated (one dose of inactive vaccine), fully vaccinated (two doses of inactive vaccine or one dose of adenovirus vaccine), and booster vaccinated (three doses of inactive vaccine or two doses of adenovirus vaccine).The two key outcomes in this study were symptom burden and disease course. Fifteen symptoms related to COVID-19 were collected, including fever (peak temperature >37.3°C), chill, sore throat, hoarse voice, cough, nasal congestion, runny nose, headache, dizziness, dyspnea, myalgia, otologic symptoms, palpitation, abdominal pain, and diarrhea. Following a previous study , we consDefining long COVID in AS is challenging due to the lack of a globally accepted definition and the overlap of symptoms between long COVID and AS, such as fatigue and arthralgia . In thist-tests or Mann-Whitney U tests for continuous variables. Missing data were addressed using multiple imputations with five iterations, assuming that the data were missing at random.In the descriptive analysis, we assessed the differences in proportions and medians of variables between the exposure group and the unexposed group using chi-squared tests for categorical variables and versus no medication use on COVID-19 symptom burden and disease course. First, to evaluate the association between COVID-19 symptom burden and baseline medication exposure, we used modified Poisson regression with a robust (sandwich) estimation of variance    to calcuriables) , smokingQuestionnaires were collected from 658 AS patients, out of which 112 patients reported negative results for COVID-19 infection. Additionally, 25 patients were taking other AS medications, including 11 patients using Secukinumab, 6 patients using Tofacitinib, and 8 patients using traditional Chinese medicines. These patients were excluded from the analysis, leaving a total of 521 patients . Among tRegarding AS treatments, 71 patients (13.6%) underwent NSAIDs monotherapy, 95 (18.2%) received NSAIDs in combination with csDMARDs, 38 (7.3%) had csDMARDs monotherapy, 232 (44.5%) were treated with TNFi, and 85 (16.3%) did not receive any of the mentioned medications. Compared to patients who reported no AS medication usage, patients who received TNFi were more likely to be men (196 [84.5%] vs 66 [77.6%]) and less likely to be older than 55 years (6 [2.6%] vs 5 [5.9%]). Patients with NSAIDs monotherapy were more likely to be overweight (38 [53.5%] vs 31 [36.5%]) and have COPD (4 [5.6%] vs 1 [1.2%]), but less likely to be older than 55 years (1 [1.4%] vs 5 [5.9%]). On the other hand, patients with csDMARDs monotherapy were more likely to be older than 55 years (5 [13.2%] vs 5 [5.9%]), but less likely to be male (26 [68.4%] vs 66 [77.6%]). Besides, patients treated with csDMARDs monotherapy, NSAIDs & csDMARDs or TNFi had lower BASDAI , 2.1  and 2.2 , respectively) and BASMI , 1.0  and 3.0 , respectively) than patients without previous medication use  for BASDAI and 4.0  for BASMI), whereas patients with NSAIDs monotherapy had worse BASFI ) and comparable BASDAI ), BASMI ) than patients without previous medication use  for BASDAI, 4.0  for BASMI and 1.2  for BASFI) .The median (IQR) number of symptoms reported by patients with no medication usage, NSAIDs monotherapy, csDMARDs monotherapy, and TNFi were 6.0 , 8.0 , 7.5 , and 7.0 , respectively . DetaileA similar analysis was conducted to evaluate the risk of experiencing more than 10 symptoms in AS patients. Patients treated with NSAIDs, csDMARDs and TNFi had similar odds of experiencing more than 10 symptoms compared to those without medication use in both univariable analysis and after adjusting for confounding factors . Likewise, COPD was associated with greater odds of experiencing more than 10 symptoms  .The median (IQR) duration of COVID-19 symptoms in patients with no medication use, NSAIDs monotherapy, csDMARDs monotherapy, and TNFi were 8.0 , 9.0 , 9.5 , and 9.0  days, respectively . The uniSimilarly, when LC28 was considered as an increased disease course in COVID-19, patients with NSAIDs, csDMARDs, and TNFi had similar odds of LC28 compared to those without medication usage, both in univariate analysis and after adjustment . Additionally, having cardiovascular disease (CVD) was found to be associated with greater odds of LC10 , but not LC28  in the population .Among the 232 patients treated with TNFi, 104 patients received TNFi monotherapy (Group 1), while 128 patients were prescribed NSAIDs or csDMARDs concomitantly (Group 2). The median (IQR) number of symptoms reported by patients in Groups 1 and 2 were 7.0  and 8.0 , respectively . After aThe median (IQR) duration of COVID-19 symptoms in Groups 1 and 2 were 9.0  and 10.0  days, respectively . In multIn this study, we examined a cohort of patients with AS and mild-moderate COVID-19. We found no association between previous use of csDMARDs or TNFi and worse COVID-19 outcomes. To our knowledge, this is the first study to investigate the impacts of csDMARDs and TNFi on AS patients with mild-moderate COVID-19. Our findings are significant as they alleviate concerns about the potential increased disease burden of COVID-19 in AS populations using csDMARDs or TNFi. These findings can potentially guide future treatment decisions.Despite the emergence of studies reporting the characteristics, outcomes and associated factors for COVID-19 in individuals with systemic autoimmune diseases , there aTo assess the disease burden of mild-moderate COVID-19, we measured the number of self-reported symptoms and the duration of recovery in patients. Obviously, higher number of symptoms and longer recovery time indicate a greater burden of illness. After accounting for potential confounding factors, patients treated with csDMARDs or TNFi had similar odds of experiencing more than five symptoms compared to those not taking AS medications. The exception was patients on NSAIDs monotherapy, which presented a subtly worsening effect on symptom burden with borderline significance . However, when we evaluated the outcome of having more than 10 symptoms, no significant association with COVID-19 symptoms burden was detected for all medications, both in univariable and multivariable analyses. Initially, there were concerns about the use of NSAIDs in the early stages of the pandemic due to their theoretical potential to worsen COVID-19 outcomes . HoweverPersistent COVID-19 symptoms, also known as “long COVID,” are widespread among individuals with COVID-19 . It is eOur study has several limitations that should be acknowledged. Firstly, there existed sampling bias due to various factors, including a single geographical area, high COVID-19 vaccination coverage (85%), a relatively young population, and limited comorbidities, which generally resulted in better COVID-19 outcomes. Secondly, in this study, patients were classified as having mild-moderate COVID-19 retrospectively. However, it should be noted that patients presenting with mild-moderate symptoms initially can progress to severe outcomes, especially among older individuals, men, and those with comorbid conditions such as cardiometabolic and pulmonary conditions . PreviouIn conclusion, our findings suggest that the use of csDMARDs or TNFi does not result in an increased symptom burden or longer recovery time in individuals with AS following a mild-moderate COVID-19 infection. This information should be taken into account when making treatment decisions between patients and physicians.


A total of 287 actinobacterial isolates affiliated to 10 genera in eight families of six orders were isolated from five different mangrove rhizosphere soil samples, specififically, Streptomyces (68.29%) and Micromonospora (16.03%), of which 87 representative strains were selected for phylogenetic analysis. The crude extracts of 39 isolates (44.83%) showed antimicrobial activity against at least one of the six tested indicator pathogens, especially ethyl acetate extracts of A-30 (Streptomyces parvulus), which could inhibit the growth of six microbes with MIC values reaching 7.8 μg/mL against Staphylococcus aureus and its resistant strain, compared to the clinical antibiotic ciproflfloxacin. Furthermore, 79 crude extracts (90.80%) and 48 (55.17%) of the isolates displayed anticancer and immunosuppressive activities, respectively. Besides, four rare strains exhibited potent immunosuppressive activity against the proliferation of Con A-induced T murine splenic lymphocyte in vitro with an inhibition rate over 60% at 10 μg/mL. Type I and II polyketide synthase (PKS) and non-ribosomal synthetase (NRPS) genes were detected in 49.43, 66.67, and 88.51% of the 87 Actinobacteria, respectively. Signifificantly, these strains  harbored PKS I, PKS II, and NRPS genes in their genomes. Nevertheless, their bioactivity is independent of BGCs in this study. Our findings highlighted the antimicrobial, immunosuppressive, and anticancer potential of mangrove rhizosphere Actinobacteria from Hainan Island and the biosynthetic prospects of exploiting the corresponding bioactive natural product.Mangrove rhizosphere soils host diverse Actinobacteria tolerant to numerous stresses and are inevitably capable of exhibiting excellent biological activity by producing impressive numbers of bioactive natural products, including those with potential medicinal applications. In this study, we applied an integrated strategy of combining phylogenetic diversity, biological activities, and biosynthetic gene clusters (BGCs) screening approach to investigate the biotechnological importance of Actinobacteria isolated from mangrove rhizosphere soils from Hainan Island. The actinobacterial isolates were identifified using a combination of colony morphological characteristics and 16S rRNA gene sequence analysis. Based on the results of PCR-detected BGCs screening, type I and II polyketide synthase (PKS) and non-ribosomal synthetase (NRPS) genes were detected. Crude extracts of 87 representative isolates were subjected to antimicrobial evaluation by determining the minimum inhibitory concentration of each strain against six indicator microorganisms, anticancer activities were determined on human cancer cell lines HepG2, HeLa, and HCT-116 using an MTT colorimetric assay, and immunosuppressive activities against the proliferation of Con A-induced T murine splenic lymphocytes  Natural products have served as powerful therapeutics against emerging infectious diseases and multidrug-resistant human pathogens since the golden age of antibiotics of the mid-twentieth century , immunosuppressive agents , anti-tumor agents , and enzymes and enzyme inhibitors . Furthermore, ~200 compounds, including some “hot biomolecules,” such as salinosporamide A , antibiotic drug leads, including xiamycins, rifamycins, and antimycin A were discovered from mangrove Actinobacteria  makes Actinobacteria well-known producers of diverse secondary metabolites , and there are no copyright issues. All samples  were collected from depths 10–15 cm from the main roots. Five parallel soil samples at each site within a 20 m2 area were mixed to obtain one sample, then packed in sterile bags and transported to the laboratory quickly. All samples were air-dried before grinding with a mortar and pestle.Five mangrove rhizosphere soil samples were collected in 2021 at different mangrove sites of Hainan Island, Hainan province, China. The locations where the samples were collected and their information are shown in −4. An aliquot of diluted suspensions  was spread in triplicate onto agar plates of the above media and incubated at 28°C for 1–4 weeks. From each plate, all phenotypically distinct colonies were picked and purified by repeating streaking using a sterile L-shaped loop onto freshly prepared ISP2 agar plates until pure isolates were obtained. Putative actinobacterial colonies were selected based on colony morphology, the color of aerial and substrate mycelium, the production of diffusible pigments, and the growth rate. The pure individual strains were cryopreserved on ISP2 agar slants at 4°C for short-term storage and as glycerol suspensions  at −80°C for long-term storage.Seven media were prepared to isolate the actinobacterial strains . All med2O. The following conditions were observed: initial denaturation at 95°C for 3 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 10 min. The amplicons were then visualized by gel electrophoresis in 1% agarose gel. The products were purified and sequenced on the ABI PRISMTM 3730XL DNA Analyzer . The obtained sequences were compared with the sequences in NCBI (http://www.ncbi.nlm.nih.gov/) and EzBioCloud (https://www.ezbiocloud.net/) databases . The water layer was lyophilized, and its residue (WF) was collected.Approximately 87 representative strains were inoculated into 100 mL ISP2 broth in 500 mL conical flasks and incubated for 7 days at 28°C with shaking (180 rpm). A total of 300 mL (3 × 100 mL) cultural broth of each strain was centrifuged at 4,200 rpm at 20°C for 20 min to separate the mycelium from the broths. The supernatants were extracted with EtOAc  three times and then diluted with HMeanwhile, the mycelia were soaked overnight in acetone and then filtered. The acetone extract was dried in vacuo to obtain mycelia fraction (MF). Currently, ethyl acetate is an efficient solvent used for extracting actinobacterial culture supernatant and is environmentally friendly. Most of the un-polar and semi-polar components were transferred from the aqueous to the organic phase, and then the extracts were easily separated from water and dried by rotary evaporation. However, it was less efficient in extracting polar substances. The mycelium was ultimately extracted using acetone due to its compatibility with un-polar, semi-polar, and polar components. All the titled extracts were dissolved in dimethyl sulfoxide  and stored at 4°C until use.Staphylococcus aureus , gram-negative Pseudomonas aeruginosa , Escherichia coli , and pathogenic Candida albicans  were used to evaluate the antimicrobial activity of the extracts from the 87 actinobacterial isolates by the serial dilution technique  was assigned to the lowest concentration that completely inhibited the growth of a given indicator microorganism. Ciprofloxacin (1 mg/mL) and amphotericin B (1 mg/mL) were used as positive controls.Six indicator microorganisms, including gram-positive 2 and 95% air at 37°C for 24 h. Subsequently, the cells were treated with 10 μg/mL crude extracts added to each well and incubated for 24 h under the above conditions. Any traces of the extracts were removed by washing with PBS and applying medium with 20 μL MTT dissolved in 100 μL medium for 4 h at room temperature. DMSO (200 μL) was added to dissolve the formazan crystals. The absorbance was measured at 570 nm by a microplate reader. Doxorubicin (10 μg/mL) was used as the positive control.The anticancer screening was performed using the 3--2,5-diphenyltetrazolium bromide (MTT) method according to our previous report  and 50 μL RPMI 1640 medium containing 40 μg/mL crude extracts were added to a final volume of 200 μL. The control group was treated with RPMI 1640 medium containing CsA or only 0.1% DMSO. After 48 h of incubation at 37°C in 5% CO2, 20 μL CCK-8 reagent was added to each well and then placed in the incubator to culture for 4 h in a dark condition. The absorbance of each solution was measured at 450 nm with a microplate reader. Cyclosporine A (10 μg/mL) was used as the positive control.Tetrazolium salt-based CCK-8 assay was used to quantify the immunosuppressive activities against the proliferation of Con A -induced T lymphocyte proliferation with some modification  K1F (5′-TSA AGT CSA ACA TCG GBC A-3′) and M6R , Simpson's Diversity Index (1-D), Pielou, Invsimpson, and Chao1 indices) were created or calculated using R software (version 3.7.0) with Ggplot2, pPheatmap, and Vegan packages, respectively. The Bray-Curtis matrix was employed to evaluate the β-diversity and visualized using Principal Coordinate Analysis (PCoA). Results were expressed as mean ± standard deviation (SD) of triplicate measurements for the antimicrobial, immunosuppressive, and anticancer activities. The relationship of biological activities and shared isolates were analyzed using the UpSetR package in R software for antimicrobial, while ordinary one-way ANOVA (p < 0.05) followed by Dunnett's t-test using Statistical Analysis System  and GraphPad Prime 7 for immunosuppressive and anticancer activities. The correlation of BGCs and representative isolates was investigated using the Venn Diagram package in R software.Bar plot, heat map, and α-diversity , followed by Micromonospora  and Curtobacterium .Based on the analyses of partial 16S rRNA sequences and phenotypic characteristics, 87 representative isolates were selected for the phylogenetic analysis utilizing the MEGA 7.0 software. The confidence level was calculated at a 1,000 bootstrap value to visualize the relatedness of the isolates and their closely related actinobacterial species using the neighbor-joining (NJ) algorithm . The actStreptomyces and Rhodococcus were dominant in S1 and S3; Curtobacterium and Microbacteriumcan were recovered in S1, while isolates belonging to Sinomonas, Gordonia, and Mycobacterium were observed in S3. The Actinomadura species were the most diverse in S5. Micromonospora could be obtained from S4, but not in abundance. Nocardia could be obtained from all samples but not in S4.The distribution of 287 representative isolates from five sampling sites is displayed in Curtobacterium, Microbacterium, and Actinomadura and could be easily isolated from M2. In contrast, Gordonia and Mycobacterium could be easily isolated from M4. Actinobacteria genus Nocardia, Micromonospora, Streptomyces, Sinomonas, and Rhodococcus showed no significant difference in the isolation media  of the total isolates. M4  and M6  were the second best, followed by M7  and M3 . However, M5 yielded the highest number of isolates with low diversity , and M1 yielded the lowest number and variety of isolates , as shown in on media .H'= 1.15, 1–D = 0.56), followed by S3 . The uneven Pielou and Invsimpson index indicated variant species composition across different sites. The observed number of actinobacterial isolates in S1 and S3 were still lower than predicted in the estimate using the non-parametric Chao1indice. All indices agreed with the less diverse communities being in S4. The principal coordinate analysis (PCoA) plot revealed differences (β-diversity) in the composition of actinobacterial communities , Shannon (0.21–1.15), Simpson (0.10–0.56), Pielou (0.30–0.67), Invsimpson (1.11–2.26), and Chao1 (2–24) varied significantly among the studied sampling sites . The Shamunities . The firH' = 1.31, 1–D = 0.67, Pielou = 0.073, Invsimpson = 3.0) to be suitable for Actinobacteria isolation from rhizosphere soil and more diverse than the others. Unconstrained PCoA showed a distinct clustering pattern, and M2, M6, and M7 clustered together, Bray-Curtis metrics explained 84.49% of the variation , Micromonospora (5), Curtobacterium (3), Actinomadura (1), Sinomonas (1), and Gordonia (1), exhibited antagonistic activity against at least one of the tested pathogen at the selected concentration of 1 mg/mL (P<0.05) (S. aureus), while one was active only against gram-negative bacteria (P. aeruginosa and E. coli), and two were active only against C. Albicans. Regarding the sensitive pathogenic indicators, the activity against S. aureus (ATCC 29213) was clearly the most frequent , and activity against P. aeruginosa was the least frequent , while 14.94% (13) of the isolates were active against E. coli (ATCC 25922). As for the resistant pathogenic indicators, activity against S. aureus (ATCC 25922) was the most frequent , compared to E.coli . Besides, 12 strains (13.79%) were active against the fungal pathogen C. albicans. Additionally, the antimicrobial activity of the same strain was significantly different (P < 0.05) in different extract fractions, but all of the antimicrobial activities were weaker than that of the positive control. EF extracts were determined to be more suitable for antibiotic production than MF and WF. EF from the actinobacterial isolates, A-30 (Streptomyces parvulus), A-326 (Streptomyces broussonetiae), J-151 (Streptomyces seoulensis), R-77 (Actinomadura geliboluensis), and ZRJ-11 (Streptomyces qinglanensis) was most potent against S. aureus and its resistant strain with MIC values reaching 7.8 μg/mL, compared to the clinical antibiotic ciprofloxacin (MIC = 3.9 μg/mL). Of them, strain A-30 showed the broadest antimicrobial spectrum against six indicator test microorganisms. A broad spectrum of antimicrobial activities was also obtained with EF of several Streptomyces isolates, A-56 (S. pseudogriseolus), A-70 (S. caniferus), A-107 (S. racemochromogenes), A-189 (S. bingchenggensis), A-326 (S. broussonetiae), ZRJ-1 (S. hygroscopicus), J-151 (S. seoulensis), and J-170 (S. corchorusii) and were active against four or five indicators. Notably, some rare actinobacterial isolates, such as A-498 (Micromonospora haikouensis) and R-77, exhibited significant inhibition toward gram-positive bacteria, which could be a potential storehouse for novel antibiotics.Crude extracts of 87 representative isolates were subjected to antimicrobial evaluation by determining the MIC of each strain against six indicator microorganisms. Approximately 39 strains (44.83%), affiliated to six genera, including (P<0.05) , 6. The in vitro. Our results showed that 48 strains (55.17%) affiliated to eight genera, Streptomyces , Micromonospora , Curtobacterium , Actinomadura , Gordonia , Sinomonas , Nocardia , and Rhodococcus , displayed different levels of inhibitory activity against T cells growth at 10 μg/mL extracts (Gordonia rhizosphera), A-272 (Micromonospora maritima), A-23 (Nocardia arthritidis), and A-259 (Curtobacterium flaccumfaciens), which showed an inhibition rate of 76.84, 69.57, 63.75, and 61.77%, respectively. EF extracts of the isolates had the highest occurrence of immunosuppressive activity compared with those of other fractions from the water phase or mycelium. Cyclosporine A was used as the positive control, and the inhibition rate was 56.08%. Notably, the 10 Actinobacteria were stronger than the inhibition rate of cyclosporine A, suggesting that these actinobacterial species could have high potential as producers of immunosuppressive compounds.Representative strains were evaluated for immunosuppressive activities against the proliferation of Con A-induced T murine splenic lymphocytes extracts , 6. AddiStreptomyces , Micromonospora , Curtobacterium , Nocardia , Sinomonas , Gordonia (2.30%), Mycobacterium , Actinomadura , Rhodococcus , and Microbacterium , suggesting the cytotoxic potential of these actinobacterial isolates. In general, 13 extracts (14.94%) were effective against all three tested cancer cell lines, most obtained from the isolates belonging to the genus Streptomyces and none from Micromonospora, Rhodococcus, Microbacterium, Actinomadura, and Mycobacterium. The majority of extracts, 78.16% (68 strains), showed cytotoxicity and displayed a HepG2 cell viability percentage ≤50%. Of these, 10 EF extracts exhibited significant cytotoxicity against HepG2 cells with an inhibition rate over 76.89%, including six rare Actinobacteria A-103 (Microbacterium paraoxydans), R-77 (Actinomadura geliboluensis), A-217 (Micromonospora carbonacea), A-529 (Micromonospora saelicesensis), A-260 (Gordonia terrae), and A-498 (Micromonospora haikouensis), with an inhibition rate of 78.67, 78.31, 77.78, 77.59, 77.05, and 76.89%, respectively. Meanwhile, 66.67% (58 strains) of the extracts resulted in cell death of more than 50% of HeLa cells, with 10 water extracts showing strong cytotoxic effects against the HeLa cell line with an inhibition rate of more than 79.38%. In particular, four rare Actinobacteria A-383 (Nocardia testacea), A-251 (Micromonospora chersina), A-136 , and A-379 (Micromonospora vinacea) showed the highest activity suppressed 82.38, 82.35, 82.02, and 81.81% of the HeLa cells proliferation. In addition, extracts of 21.84% (19 strains) tested and exhibited cytotoxic effects against HCT-116 cells, among which 7 EF extracts could significantly antiproliferative on HCT-116 cells with an inhibition rate over 61.22%, while including the rare actinobacterial strain A-259 . Doxorubicin was used as the positive control, and the inhibition rate were 79.93%, 80.85%, and 61.74% for HepG2, HeLa, and HCT-116 cells, respectively. Notably, EF extracts of two Streptomyces species (A-119 and A-29) and WF extracts of five isolates  were stronger than the cytotoxicity of doxorubicin toward HepG2 and HeLa, respectively, suggesting that these seven actinobacterial species could have high potential as producers of anticancer compounds.The anticancer activities of the actinobacterial crude extracts were determined on human cancer cell lines HepG2, HeLa, and HCT-116 using an MTT colorimetric assay. Out of the 87 isolates, 79 strains (90.80%) tested inhibited the cancer cell's proliferation to some extent. They exhibited a percentage of viability cell value ≤50% at a concentration of 10 μg/mL extracts , 6. ThesStreptomyces, PKS-I/II and NRPS genes were seldom detected at the same time in rare Actinobacteria studied except for two strains, A-635 (Micromonospora humi) and A-252 (Rhodococcus hoagii).To investigate the mangrove rhizosphere soil-derived actinobacterial isolates for their potential to produce secondary metabolites, we detected the conserved regions of BGCs through PCR-based screening targeting the KS domain of PKS-I, KSα domain of PKS-II and AD domain of NRPS. In total, 86 (98.90%) of 87 strains showed an unambiguous amplicon band of the expected size in the agarose gel for at least one of three targeted genes , 5. MoreStreptomyces isolates were distinctively separated into different groups. Among the 39 active strains, 14 isolates (35.90%) had NRPS, type I and type II PKS (Cluster 1), 10 isolates (25.64%) lacked type I PKS (Cluster 4), six isolates (15.38%) lacked type II PKS (Cluster 3), one isolate lacked NRPS (Cluster 2), and eight isolates (20.51%) had only contain one type of biosynthetic sequences (Clusters 5–7). Interestingly, active isolates that harbored all three genes (Cluster 1) expressed different bioactivity strengths. A-369 (Streptomyces prasinosporus) and J-33 (S. griseoruber) were only active against one pathogen. On the contrary, A-30 (Streptomyces parvulus) showed the broadest antimicrobial spectrum against six indicator test microorganisms. Furthermore, 12 isolates (Cluster 8) did not elicit antimicrobial activity against six tested pathogens despite their genomes harboring type I and type II PKS and NRPS.With the confirmation of multiple BGCs in 86 of the representative 87 isolates, we hypothesized that bioactivities were positively correlated with the number of BGCs present in its genome. Thus, type I and II PKS and NRPS were associated with active and inactive actinobacterial isolates. Relationships formed were visualized through “Gephi.” The network visualization showed that 87 mangrove rhizosphere actinobacterial isolates aggregated into 14 major clusters based on their identified BGCs and antimicrobial activity, regardless of the number of susceptible pathogens . We obseSinomonas halotolerans; Cluster 8) lacked all BGCs and yet were effective against all of the three tested cancer cell lines and all eight inactive stains (Clusters 9–12) harboring at least one type of BGCs were devoid of any anticancer activity.Similarly, a relationship analysis between anticancer activity and BGCs of 87 rhizosphere actinobacterial isolates showed aggregation of twelve major clusters . Even 23Furthermore, the relationship analysis of 87 rhizosphere actinobacterial isolates' immunosuppressive activity and BGCs showed different clusters of active and inactive strains . The 48 Streptomyces and Micromonospora still accounted for the predominant numbers of the isolates, a finding in line with previous studies. For instance, Zhao et al. , followed by S3 (sampled surrounding Rhizophora apiculata in March 2021). All indices agree with the less diverse communities being in S4 (sampled surrounding Rhizophora stylosa in January 2021). This may be because Actinobacteria present in the rhizosphere soil can be transferred easily between the plant roots, allowing the plant to determine the composition of its bacterial community  and the genera microbacterium on Gauze's Medium No. 2 (M2). However, < 5% of all known Actinobacteria were readily cultivable using conventional culture-based techniques, and the rest remain unexplored  is useful as a guide to select strains from a specific genus or species of particular interest or even to discard redundant isolates or those pathogenic strains from a collection. Nevertheless, this taxonomical data alone does not provide biological or metabolic information  and represents the largest group of biomolecules  and WF extracts of A-326 were more potent than the cytotoxicity of doxorubicin toward HepG2 and HeLa; EF of six Streptomyces species, J-167, A-57, A-43, J-71, A-261, and ZRJ-2 exhibited potent immunosuppressive activity against the proliferation of Con A-induced T murine splenic lymphocytes.Our results showed that EF of the Salinispora tropica CNB-392 and is currently in phase III clinical trials for treating a series of different cancer types , was isolated from a mangrove-derived Streptomyces albidoflavus I07A-01824  and R-77 (Actinomadura geliboluensis), anticancer strains A-383 (Nocardia testacea), A-251 (Micromonospora chersina), A-136 , and A-379 (Micromonospora vinacea), and immunosuppressive strains A-570 (Gordonia rhizosphera), A-272 (Micromonospora maritima), A-23 (Nocardia arthritidis), and A-259 (Curtobacterium flaccumfaciens). EF of the J-140  exhibited antimicrobial activity, showing the strengths of this multiple biological screening approach to guide isolation efforts on unknown compounds and the capabilities to identify new sources of known active compounds. The strong inhibitory activities of these strains against a variety of pathogens, cancer cells, and Con A-induced T murine splenic lymphocytes are a good indicator that these isolates could be potential candidates for the production of corresponding bioactive secondary metabolites. Four rare strains, A-136 , A-252 (Rhodococcus hoagii), A-260 (Gordonia terrae), and A-290 (Curtobacterium oceanosedimentum) showed good antitumor activity in three tumor cells. Recently, mangromicins, a group of new secondary metabolites with unique chemical structures, were found from Lechevalieria aerocolonigenes K10-0216 isolated from a mangrove sediment sample by Omura s group  detected in this study was relatively low compared to the percentage of isolates with at least one type of biosynthetic sequences (98.90%). Most of the isolates showed the presence of genes that encoded PKS/NRPS enzymes, which also showed antimicrobial, anticancer activity, or immunosuppressive capacity. However, eight inactive strains harbored at least one type of BGCs, whereas isolate A-136 lacked all BGCs and yet exhibited significant anticancer activity. Twelve isolates with none or only one BGC present still displayed different levels of immunosuppressive activity. The low bioactivity correlation with biosynthetic gene clusters of the isolates was consistent with the findings of a subset of the researchers  approach, developed by Zeeck in the early 2000's, has been demonstrated to be a simple and effective method (Bode et al., Streptomyces-Micromonospora” as the major group and demonstrated high biosynthetic potential. Using cues from the current investigation, these biosynthetic Actinobacteria derived from mangrove rhizosphere soil, along with those that displayed bioactivity, should be given priority for the exploration of the corresponding structurally interesting biological natural products.In conclusion, this study demonstrated that Actinobacteria from mangrove rhizosphere soil in Hainan Island are underexplored sources of antimicrobial, anticancer, and immunosuppressive agents concerning their metabolic potential for polyketides and non-ribosomal peptides. A total of 287 culturable actinobacterial isolates of at least 10 genera within 8 families of 6 orders were revealed. Of 87 representative strains selected for biological activities, the PKS/NRPS screening indicated “The original contributions presented in the study are included in the article/JX conceived the study, designed the experiments, did the final revision of the manuscript, supervision, project administration, and funding acquisition. R-jZ and N-lW collected the samples. J-jY and R-jZ did the phylogenetic diversity, biosynthetic gene cluster screening, formal analysis, and data curation. R-jZ did the antimicrobial assay. D-dZ and D-dC performed the anticancer assay. X-lD and R-jZ did the immunosuppressive assay. J-jY did validation, writing of the original draft, and visualization. All authors have read and agreed to the published version of the manuscript.


Calponin 2 (CNN2) is a prominent actin stabilizer. It regulates fatty acid oxidation (FAO) by interacting with estrogen receptor 2 (ESR2) to determine kidney fibrosis. However, whether CNN2 is actively involved in acute kidney injury (AKI) remains unclear. Here, we report that CNN2 was induced in human and animal kidneys after AKI. Knockdown of CNN2 preserved kidney function, mitigated tubular cell death and inflammation, and promoted cell proliferation. Distinct from kidney fibrosis, proteomics showed that the key elements in the FAO pathway had few changes during AKI, but we identified that 3-hydroxymethylglutaryl-CoA synthase 2 (Hmgcs2), a rate-limiting enzyme of endogenous ketogenesis that promotes cell self-renewal, was markedly increased in CNN2-knockdown kidneys. The production of ketone body β-hydroxybutyrate and ATP was increased in CNN2-knockdown mice. Mechanistically, CNN2 interacted with ESR2 to negatively regulate the activities of mitochondrial sirtuin 5. Activated sirtuin 5 subsequently desuccinylated Hmgcs2 to produce energy for mitigating AKI. Understanding CNN2-mediated discrete fine-tuning of protein posttranslational modification is critical to optimize organ performance after AKI.  CNN1, CNN2, and CNN3  is a calcium-binding protein. It has 3 isoforms encoded by homologous genes in vertebrates, and CNN3 , 5. In cAKI is a refractory clinical syndrome that refers to sudden kidney function loss within a few hours or days. Thus far, there are no effective strategies to predict, prevent, or mitigate AKI, though the field has a consensus that kidney tubules are the epicenter of damage after AKI. Tubule repair requires high energy that depends on metabolic processes to generate ATP. Consequently, the extent of tubule repair is highly associated with AKI prognosis. During tubule repair, neighboring fibroblasts emerge as orchestrators of a favorable kidney local microenvironment (KLM) . In CKD,Ketone bodies are 3 small water-soluble lipid molecules  mainly produced in the liver by breaking down fatty acids . They de+ fibroblasts/pericytes after AKI . To establish the clinical relevance of CNN2 expression and human AKI, we performed immunohistochemical staining for CNN2 in kidney biopsy specimens from patients with AKI. Compared with the expression in the nontumor normal human kidneys, CNN2 was upregulated in the kidney interstitium in patients with AKI  staining, we identified the number of TUNEL-positive tubular cells was decreased in ShCNN2 mice compared with vehicle in the 2 AKI models . WesternRantes, IL-6, and TNF-α, in ShCNN2 kidneys after AKI (+ monocytes (+ macrophages (+ or α–smooth muscle actin–positive (α-SMA+) fibroblasts/pericytes’ early activation in the kidney after AKI  between ShNC and ShCNN2 mice after IRI. Compared with ShNC kidneys, 685 and 322 proteins were up- and downregulated in ShCNN2 kidneys , IL-6, and TNF-α mRNA levels at 1 day after IRI  . Conversfter IRI . Consist kidneys . These rHmgcs2 gene transcription  with CNN2 siRNA . ELISA sFurther, we applied human CNN2 recombinant protein to treat tubular cells at different dosages. Western blot assay demonstrated that compared with CNN2-deprived CM, CNN2 protein repressed sirt5 and Hmgcs2 under hypoxic stress . Co-immuEnergy metabolism, such as glucose, fatty acid, and amino acid metabolism, is an indispensable process for maintaining cell homeostasis. Research over the past several decades has expanded our knowledge of energy metabolism in many disease-oriented research fields, such as diabetes, obesity, cardiology, and cancer. This metabolic process has evolved to have multiple pathways that respond to various pathologic stresses by generating energy. In recent years, along with the reported beneficial effects of sodium-glucose cotransporter-2 inhibitor treatment on kidneys , 21, elu+ macrophages in the ischemic kidney . All patients included in the presented study had signed the informed consent forms before they underwent kidney biopsy or nephrectomy. All studies involving human kidney sections were approved by the Institutional Review Board at the University of Pittsburgh and the University of Connecticut School of Medicine.Data are available in the YG and DZ conceived the project. YG and DZ wrote and revised the manuscript. YG, ZP, PG, and Yuanyuan Wang performed most in vivo, ex vivo, and in vitro studies involving mRNA expression analysis; Western blotting assay; immunostaining; and imaging. YG, HL, YP, and SL performed bioinformatic analysis. YG, GH, DLK, Yanlin Wang, SIB, YL, and DZ edited the manuscript. DZ supervised the entire project.


Prostate cancer (PCa) is the second most common type of cancer and the fifth leading cause of cancer-related death in men. Androgen deprivation therapy (ADT) has become the first-line therapy for inhibiting PCa progression; however, nearly all patients receiving ADT eventually progress to castrate-resistant prostate cancer. Therefore, this study aimed to identify hub genes related to bicalutamide resistance in PCa and provide new insights into endocrine therapy resistance.The data were obtained from public databases. Weighted correlation network analysis was used to identify the gene modules related to bicalutamide resistance, and the relationship between the samples and disease-free survival was analyzed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed, and hub genes were identified. The LASSO algorithm was used to develop a bicalutamide resistance prognostic model in patients with PCa, which was then verified. Finally, we analyzed the tumor mutational heterogeneity and immune microenvironment in both groups.LUC7L3, SNRNP70, PRPF3, LUC7L, CLASRP, CLK1, CLK2, U2AF1L4, NXF1, and THOC1) and 13 in the yellow module . The prognostic model composed of RNF207, REC8, DFNB59, HOXA2, EPOR, PILRB, LSMEM1, TCIRG1, ABTB1, ZNF276, ZNF540, and DPY19L2 could effectively predict patient prognosis. Genomic analysis revealed that the high- and low-risk groups had different mutation maps. Immune infiltration analysis showed a statistically significant difference in immune infiltration between the high- and low-risk groups, and that the high-risk group may benefit from immunotherapy.Two drug resistance gene modules were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that both modules are involved in RNA splicing. The protein–protein interaction network identified 10 hub genes in the brown module In this study, bicalutamide resistance genes and hub genes were identified in PCa, a risk model for predicting the prognosis of patients with PCa was constructed, and the tumor mutation heterogeneity and immune infiltration in high- and low-risk groups were analyzed. These findings offer new insights into ADT resistance targets and prognostic prediction in patients with PCa. In 2022, approximately 1,414,259 new cases of PCa were diagnosed worldwide, and 375,304 deaths were reported  activity and tumor invasion in androgen-responsive PCa and has been widely used in clinical practice . Over tiWith the advent of high-throughput sequencing and bioinformatics, researchers can classify and analyze a large number of samples, explore tumor characteristics and heterogeneity, and find new personalized markers. Based on these methods, hub genes associated with PCa progression and recurrence and CRPC have been identified , 11. HowIn the present study, our purpose is identifying hub genes related to bicalutamide resistance in PCa. These genes may be related to endocrine therapy resistance in patients with PCa and may be potential targets for reversing such resistance. Later we constructed a risk model to predict the prognosis of patients with PCa based on samples and analyzed tumor mutational heterogeneity and immune infiltration in high- and low-risk patient groups. Altogether, our findings provide new insights into ADT resistance and prognostic prediction in patients with PCa, which will help establish personalized treatment regimens and drug choice.22.1https://xena.ucsc.edu/). After excluding samples without disease-free survival (DFS), Gleason score, and T stage, 483 samples were included in this study. RNA-seq data were converted to transcripts per million to remove the effect of sequencing depth. TCGA-PRAD single nucleotide mutation data were downloaded from the Genomic Data Commons - The Cancer Genome Atlas website . A total of 1832 differentially expressed genes in PCa were obtained from the GEPIA2 database (http://gepia.cancer-pku.cn/detail.php?clicktag=degenes).Transcriptome and clinical data from The Cancer Genome Atlas - Prostate Adenocarcinoma (TCGA-PRAD) dataset were downloaded from the Xena database (2.2The pRRophetic package  was used2.3http://metascape.org/gp/index.html#/main/step1). Concomitantly, the key hub genes in these modules were identified via the protein–protein interaction network (PPI). These key hub genes were identified with the MCODE plugin of Cytoscape, and statistical significance was set at p < 0.05.GO and KEGG analyses of the brown (218) and yellow modules (163) were annotated using the Metascape  website 2.4To further determine the prognostic model for drug resistance genes, univariate Cox regression analysis was used for all genes in the brown and yellow modules, and 89 prognostic genes were identified. We included these genes in the prognostic model, which consisted of 12 genes and was constructed using the LASSO algorithm. The risk score was calculated as (risk coefficient × gene expression level). Subsequently, the samples were divided into a training set and a validation set at a 1:1 ratio, and the low- and high-risk groups were divided by the median risk score. The Kaplan–Meier (KM) curve was used to describe the DFS of the low- and high-risk groups, and statistical significance was set at p < 0.05. Receiver-operating characteristic (ROC) curves were used to demonstrate the predictive efficacy of the training and validation sets for 1, 3, and 5 years.2.5The single-nucleotide mutation data of TCGA-PRAD were processed using the maftools package . The 10 2.6https://www.immport.org/home). The ssGSEA algorithm  of bicalutamide for each sample in the TCGA-PRAD dataset. WGCNA was constructed based on 1832 differentially expressed genes in PCa. A total of 434 samples were included in subsequent analyses, while 49 were excluded  are hub genes of the brown module , and the training cohort was divided into low- and high-risk groups according to the median risk score (121:121) , while the TTN mutation rate was significantly lower (8% vs. 14%). The high-risk group also presented mutations in KMT2D, FOXA1, and RYR1 . Conversely the low-risk group presented MUC16, SYNE1, and FOXA1 mutations . In summary, genomic heterogeneity was identified between the low- and high-risk populations.To determine the heterogeneity between the high- and low-risk groups in patients with PCa, we studied the mutation landscape diagram of the two groups. By displaying the 10 most mutated genes in the identified PRAD database, we observed a significantly different landscape between the high- and low-risk groups  activates chaperone genes, microtubule-based movement, positive regulation of voltage-gated potassium channel activity, macroautophagy, protein modification by small protein conjugation, and HSV-1 infection. Previous studies have found that many aberrant mRNA splice variants are upregulated in PCa, which further aggravates the disease by promoting proliferation, metastasis, tumor growth, anti-apoptosis, and drug resistance . Selectit in PCa . Nguyen ith CRPC . Tokarz  therapy . HSV-1 i therapy . TherefoLUC7L3, SNRNP70, PRPF3, LUC7L, CLASRP, CLK1, CLK2, U2AF1L4, NXF1, THOC1, PNN, PPWD1, SRRM2, DHX35, DMTF1, SALL4, MTA1, HDAC7, PHC1, ACIN1, HNRNPH1, DDX17, and HDAC6). HNRNPH1 knockdown has been shown to reduce the expression of AR and its splice variant AR-V7 (or AR3). Small interfering RNA silencing of HNRNPH1 sensitizes PCa cells to bicalutamide and inhibits prostate tumorigenesis in vivo (THOC1 (PNN (MTA1 (DDX17 (CLK1 (DMTF1 expression is related to increased cisplatin resistance in breast cancer (We performed PPI analysis of the two resistance-related gene modules separately, and identified 23 hub genes ( in vivo . HDAC6 d in vivo . High exo (THOC1 , PNN (32NN (MTA1 , DDX17 (1 (DDX17 , and CLKt cancer . TherefoRNF207, REC8, DFNB59, HOXA2, EPOR, PILRB, LSMEM1, TCIRG1, ABTB1, ZNF276, ZNF540, and DPY19L2. According to the median risk score, the patients were divided into high- and low-risk groups. The results showed that the high-risk group had a higher number of recurrences or deaths and shorter survival than the low-risk group. A high expression of these 12 genes is related to a worse prognosis. RNF207 was found to predict lymph node involvement in patients with obesity and endometrial cancer (REC8 can promote tumor migration, invasion, and angiogenesis in hepatocellular carcinoma (PILRB (TCIRG1 (ABTB1 expression via miR-4319 can inhibit colorectal cancer progression (ZNF276 can promote the malignant phenotype of breast cancer by activating the CYP1B1-mediated Wnt/β-catenin pathway (Patients with PCa with castration resistance present a shorter survival time and higher risk of progression . For surl cancer . By targarcinoma . PILRB (a (PILRB  and TCIRgression . ZNF276  pathway . SubsequSPOP and TTN in the high- and low-risk groups, respectively, which may indicate that patients with a high SPOP mutation rate have a worse prognosis, whereas those with a high TTN mutation rate have a relatively better prognosis. TP53 was the second most frequent mutation in both groups. SPOP mutations can promote PCa progression by promoting autophagy and Nrf2 activation (SPOP mutation can increase the sensitivity of PCa cells to ADT (SPOP mutations and/or CHD1 deletions are more sensitive to abiraterone treatment (SPOP mutations lead to PCa resistance to cellular stress induced by chemotherapeutic agents such as docetaxel (TP53 is the most prominent gene in pan-cancer studies, and its somatic alterations are independently associated with the rapid emergence of drug resistance in patients with metastatic CRPC (TP53 and RB1 can transform PCa AR-dependent luminal epithelial cells into AR-independent basal-like cells, which are resistant to ADT (Tumor mutation burden is closely associated with tumor heterogeneity . To detetivation . Howevers to ADT . Studiesreatment . Notablyocetaxel . TP53 istic CRPC . Mutatiot to ADT . These gFurther research, has shown that the tumor immune microenvironment is closely associated with endocrine therapy resistance . TherefoAfter immune checkpoint expression analysis, we found that CD200, CD200R1, CD70, CD80, CD86, HAVCR2, LAG3, LAIR1, LGALS9, NRP1, PDCD1, PDCD1LG2, TIGIT, TNFRSF18, TNFRSF25, and TNFSF14 were highly expressed in the high-risk group. Based on the inflammation and immune imbalance in various tumor microenvironments, the CD200–CD200R pathway is differentially regulated . In livein vivo and in vitro experiments are required, and the function of drug resistance genes needs to be further explored.Notably, this study has some strengths. Firstly, 23 hub genes associated with bicalutamide resistance were identified. These genes may be potential targets for reversing bicalutamide resistance or even other endocrine therapies resistance in PCa. Then, we constructed a prognostic model consisting of 12 genes, showing a high predictive value. Besides, we found differential tumor mutation burden and immune status in high- and low-risk groups. Therefore, our study has greater clinical implications for the prognostic assessment and selection of treatment options for patients with PCa. Yet, our study presents certain limitations. First of all, it’s a retrospective study with relatively small sample sizes in the training and test cohorts, so further studies with larger cohorts are necessary to confirm our results. Moreover, 5In summary, using public databases, we identified genes and hub genes associated with bicalutamide resistance in PCa. These genes may be related to endocrine therapy resistance in PCa, and may be potential targets for reversing endocrine therapy resistance. Concomitantly, we constructed an effective risk model to predict the prognosis of patients with PCa and analyzed tumor mutation heterogeneity and immune infiltration in high- and low-risk groups. In conclusion, this study provides new insights for the exploration of ADT resistance targets and prognosis prediction in patients with PCa, which will help establish personalized treatment options and drug choice.https://xena.ucsc.edu/, the Xena database https://portal.gdc.cancer.gov/, the Genomic Data Commons - The Cancer Genome Atlas http://gepia.cancer-pku.cn/detail.php?clicktag=degenes, the GEPIA2 database.Publicly available datasets were analyzed in this study. This data can be found here: YL and XL designed the study. YL, YP, and ZZ performed the bioinformatics analysis. HW, HZ, and MX wrote the manuscript. SW and XL supervised the study. All authors have read and approved the final manuscript. All authors contributed to the article and approved the submitted version.


In November 2021, the SARS-CoV-2 Omicron variant of concern has emerged and is currently dominating the COVID-19 pandemic over the world. Omicron displays a number of mutations, particularly in the spike protein, leading to specific characteristics including a higher potential for transmission. Although Omicron has caused a significant number of deaths worldwide, it generally induces less severe clinical signs compared to earlier variants. As its impact on blood platelets remains unknown, we investigated platelet behavior in severe patients infected with Omicron in comparison to Delta.Clinical and biological characteristics of severe COVID-19 patients infected with the Omicron (n=9) or Delta (n=11) variants were analyzed. Using complementary methods such as flow cytometry, confocal imaging and electron microscopy, we examined platelet activation, responsiveness and phenotype, presence of virus in platelets and induction of selective autophagy. We also explored the direct effect of spike proteins from the Omicron or Delta variants on healthy platelet signaling.Severe Omicron variant infection resulted in platelet activation and partial desensitization, presence of the virus in platelets and selective autophagy response. The intraplatelet processing of Omicron viral cargo was different from Delta as evidenced by the distribution of spike protein-positive structures near the plasma membrane and the colocalization of spike and Rab7. Moreover, spike proteins from the Omicron or Delta variants alone activated signaling pathways in healthy platelets including phosphorylation of AKT, p38MAPK, LIMK and SPL76 with different kinetics.Although SARS-CoV-2 Omicron has different biological characteristics compared to prior variants, it leads to platelet activation and desensitization as previously observed with the Delta variant. Omicron is also found in platelets from severe patients where it induces selective autophagy, but the mechanisms of intraplatelet processing of Omicron cargo, as part of the innate response, differs from Delta, suggesting that mutations on spike protein modify virus to platelet interactions. Several sublineages of the Omicron variant are now in circulation.The acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the coronavirus disease 2019 (COVID-19), undergoes genetic mutations leading to the appearance of new variants , it has post-mortem samples has revealed platelet-rich thrombi at sites of infected pulmonary endothelium , the best documented receptor for SARS-CoV-2, in platelets, it has been demonstrated that platelets from COVID-19 patients contain viral particles , 22, 23 pathways , 20. In pathways , 25. SARpathways , 26.Omicron variant, characterized by several mutations in the spike protein, including the receptor-binding domain, exhibits increased transmissibility potential and generally induces milder disease severity , 27, 28.Whether the Omicron variant affects blood platelets remains poorly known. Here, platelet parameters including activation and sensitivity were investigated as well as the presence of virus and the autophagy response in severe COVID-19 patients infected with different variants.22.1th, 2021 to May 25th, 2022. Eligible patients were hospitalized for hypoxemic pneumonia caused by SARS-CoV-2 infection. Variants identification was performed through complete sequencing of the spike gene. Ethical approval was obtained from the Comité de protection des personnes du sud-ouest et outre-mer (CPP2020-04-042a/2020-A00972-37/20.04.08.64705) and all patients gave their consent to participate, in accordance with the Declaration of Helsinki. Patients with malignant blood diseases or hemostatic disorders, pregnant women, minors or individuals under protective measures were excluded. Baseline characteristics including hemostasis parameters , and outcomes were recorded upon admission to the ICU. Blood was collected 3.8 ± 3.2 and 3.2 ± 1.4 days after ICU admission and 15.6 ± 10.1 and 9.1 ± 4.4 days after symptom onset for patients infected with the Delta and the Omicron variants, respectively. SARS-CoV-2 serology was assessed using the SARS-CoV-2 multi-antigen serology module  allowing detection of IgG antibodies in plasma by Simple Western capillary electrophoresis  from April 12Platelet function tests were not realized if acetyl salicylic acid or corticosteroids were taken within the 10 days prior inclusion.The recruitment of healthy donors was approved by the Toulouse Hospital Bio-Resources biobank, registered with the Ministry of Higher Education and Research (DC2016-2804). These donors were tested negative for PCR at inclusion, and had not taken antiplatelets or steroids within the 10 days before blood sampling. The cohort consisted of 50% females, with median age of 38 years .2.2® (equine) suspension was purchased from Takeda . Anti-CD62P conjugated FITC, anti-CD63 conjugated FITC, anti-PAC1 conjugated FITC, antibodies were obtained from BD Biosciences and the anti-GPVI antibody was obtained from Biocytex . The SARS-CoV-2 (2019-nCoV) Spike S1(D614G)-His recombinant protein and SARS-CoV-2 BQ.1.1 (Omicron) Spike S1+S2 trimer proteins were sourced from SinoBiological . All other reagents were purchased from Sigma Aldrich . Collagen-related peptide (CRP) was obtained from Prof. R. Farndale Laboratory , and the collagen reagent Horm2.32 at 2x108 platelets/mL for confocal microscopy and western-blot analysis.Whole blood samples were collected using Vacutainer tubes containing 3.2% citrate (Becton Dickinson). Platelet-rich plasma (PRP), platelet-poor plasma (PPP), and washed platelets were obtained through differential centrifugation as described previously . PPP, us2.4-1) at 37°C using siliconized glass cuvettes. The thrombosoft 1.6 software  was used to determine the maximal platelet aggregation percentage.Platelet aggregation was monitored with a turbidimetric method  following previously described protocols . PRP was2.5Flow cytometry was performed using FACSVERSE (BD Biosciences), and FASCsuites software (BD Biosciences) was used for data analysis. Platelet granule secretion was evaluated by measuring surface expression of CD62P and CD63, which serve as markers for α and δ granules, respectively. The activation status of GPIIbIIIa was assessed using PAC1 antibody. PRP was incubated in non-shaking conditions with TRAP at a concentration of 50 μM, U46619 at 5 µM, or CRP at 9 µg/mL for a duration of 10 minutes. Subsequently, PRP was incubated with the appropriate antibodies at room temperature for 15 minutes. After the addition of phosphate buffer saline (PBS), the samples were subjected to cytometer analysis. The results were expressed as median fluorescence intensity (MFI). The number of copy of GPIa, GPIb, GPIIIa, and GPVI on the platelet surface was quantified using a specific commercial kit  following the manufacturer’s instructions .2.6The proinflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and TNF alpha were quantified in human plasma using micro-ELISA using ELLA technology (ProteinSimple). Soluble P-selectin, and soluble CD40L were measured in human plasma using ELISA kits  following the manufacturer’s instructions. The capillary western blot assay was performed using all reagents provided by ProteinSimple, except for the primary antibodies, which were obtained separately. Platelet homogenates were prepared and analyzed as previously described .For western blotting, washed platelets from patients or controls were subjected to analysis using specific antibodies according to standard procedures.2.7Platelets in 1 mL of PRP were fixed for 1 hour at 37°C with 2.5% glutaraldehyde in 19 ml of 0.1 M Sorensen phosphate buffer (pH 7.4). TEM was performed and data analyzed as previously described .2.88/mL) were prepared and processed as described previously (Platelets (2.10eviously . Confocaeviously .2.98 platelets/mL) were incubated in polypropylene tubes with or without  spike proteins from Delta or Omicron variants , for 1, 10, and 30 minutes at 37°C without stirring. Platelets were then analyzed as we recently described  was used to realize the figures.33.1th, 2021 to May 25th, 2022 infected with SARS-CoV-2 Delta variant (n=11) or Omicron variant (n=9), as assessed by RT-PCR and spike gene sequencing, were enrolled as well as 10 healthy donors. The biological and clinical data of the patients are provided in vs. 125 (68–235) p<0.03] at admission (Twenty patients admitted to the ICU from April 12dmission Table 1.Table 1Platelet activation markers and reactivity in patients with SARS-CoV-2 Omicron Variant.3.2IIbβ3) and GPVI significantly decreased in platelets from severe patients infected with Omicron, suggesting the occurrence of a shedding process  and CD63, respectively, as well as the activation of αIIbβ3 integrin, did not show significant differences in controls and in patients infected with the Omicron variant Table 1.3.3Infection has been shown to modify the transcriptome of megakaryocytes and platelets, leading to the induction of an antiviral response phenotype . For exaPlatelets from six severe Omicron-infected patients were analyzed by TEM. Characteristic crown-like structures exhibiting a size consistent with SARS-CoV-2 particles were observed .A distinguishing feature of platelets from severe Delta-infected subjects was the existence of enlarged vesicles that were distinct from the open canalicular system (OCS) . Electro3.4We have previously demonstrated that platelets can activate an intrinsic antiviral defense mechanism involving the degradation of viral material through autophagosomes and autophagolysosomes . TEM anaSuper-resolution microscopy showed a weaker colocalization between the spike protein and LC3B in platelets from Omicron-infected patients, compared to Delta. The colocalization between spike protein and EEA1 (early endosome marker) was relatively low and similar, while the colocalization between spike protein and Rab7 (a late endosome marker) was significantly increased in platelets from Omicron-infected patients , the str3.5Subsequently, we investigated whether the differences in the intraplatelet processing of the two variants would correspond to distinct signaling potential of their respective spike proteins. Washed control platelets were incubated for 1, 10 and 30 minutes with the two spike proteins in non-shaking conditions, and the phosphorylation of signaling intermediates was assessed by phosphoflow cytometry . Both sp4The involvement of platelets in severe COVID-19 has been established in various studies and patient cohorts worldwide , 33, 34.In our cohort of patients, with similar comorbidities, we observed that the overall severity, as assessed by the clinical-biological SAPS II score, was more unfavorable for the Delta variant. Our study indicates that patients infected with Delta had a worse outcome compared to those infected with omicron. This is reflected in multiple organ failures, higher incidence of mechanical ventilation and longer ICU and hospital stays in the Delta patients group. However, it is noteworthy that the criteria for ICU admission were broader during the period of Delta variant patients, which may have contributed to higher average PaO2/FiO2 ratios at the time of admission. Nonetheless, these patients remained severe, as evidenced by higher SAPS II scores and longer lengths of stay compared to the Omicron variant.IIbβ3 and GPVI, related to proteolytic cleavage during platelet activation, was also observed. These preactivation platelet phenotypes have already been reported in severe subjects infected with the Delta variant , decreased number of microtubules and increased pseudopodia. Shedding of platelet surface markers including GPIaIIa, α variant , 19, 22.nfection , 42. Thunfection . Howevernfection , 34. Planfection –9. We fonfection , 26. Of nfection . Interesnfection .We also observed that, while IRAK4, a reflection of TLR activation, was upregulated and phosphorylated in platelets from patients infected with Delta, only weak upregulation and phosphorylation were observed in platelets from subjects with Omicron. This result suggests that, as previously observed for HIV-1 pseudovirions , infectiIn vitro, the absence of TMPRSS2 in host cells redirects SARS-CoV-2 Delta variant toward an endocytic pathway involving Rab7-positive late endocytic vesicles . The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.Conception-design of the work: BP, CG, and FV-B. Patient inclusion, clinical management duties, and ethical approvals: FV-B, BC, AR, and SV. Experimental design and execution of the majority of the experiments: CG and BC. Data compilation, analysis, and interpretation: CG, AR, SV, FV-B, and BP. Manuscript writing: BP, CG, and FV-B. Manuscript review and critical editing: all authors. All authors contributed to the article and approved the submitted version.


Serum levels of inflammatory cytokines such as IL-6, high-sensitivity C-reactive protein as well as uric acids have been considered as predictors of severe outcomes in patients with coronary artery disease (CAD) associated with hypertension (HTN) and Type2 Diabetes Mellitus (T2DM). Anthropometric parameters were recorded and measured the levels of major risk factors in 80 patients with hypertension, coronary artery disease with and without Type 2 diabetes mellitus and 40 healthy controls were included.Comparison were performed by classifying the study subjects into 3 groups namely- Group I: Controls (n=40); Group II: HTN, CAD without T2DM(n=40); and Group III: HTN,CAD with T2DM(n=40).The values of BMI, weight, SBP, DBP were greater in patients with T2DM CADgroup when compared to without diabetes group. Data shows that there is a positive correlation between IL-6, hs-CRP and uric acid concentrations and are statistically significant. The evaluated high levels of inflammatory cytokines and uric acids in hypertensiveCAD patients with diabetes might be helpful in the diagnosis of patients at greater risk. Coronary artery disease is the most frequent acute and chronic condition in the industrialised world, and it is the main cause of mortality and morbidity. The cardiovascular epidemic is a global phenomenon that accounts for about half of all deaths indeveloped countries. There had been 2.03 million fatalities in India in the year 2010 owing to CAD, with the prevalence of CAD being 2-3 times greater in urban than rural populations, with 96.7 percent per 1000 people in urban and 27.1 percent per 1000population in rural . Higher The study comprised of 120 subjects involving both males and females. The subjects were classified into three groups: Group 1: Controls (n=40), Group II: HTN, CAD without T2DM (n=40), and Group: III HTN, CAD with T2DM (n=40). The baseline characteristicssuch as age, gender, height, weight, BMI, waist-hip ratio, alcohol consumption, SBP, DBP, and smoking were collected. The inclusion criteria for selecting the subjects for the study were the age of the subjects were preferably between 30 and 70 years, Boththe groups of patients with CAD proven by Angiogram. The exclusion criteria were patients who had history of autoimmune disease, liver disease, kidney diseases, other type of diabetes mellitus, Thyroid diseases, congenital heart diseases, other types of heartdiseases, and also persons whoever were not willing to participate in the study were excluded. The study was conducted in PES medical college super specialty hospital, Kuppam, Andhra Pradesh. The study participants were recruited after approved by InstitutionalEthics committee IEC Ref no: PESIMSR/IHEC/52/2017-18), and an informed consent form.10ml of and 3ml of postprandial venous blood samples were collected from the cubital fossa from all the participants after 8 to 10 hours of fasting. Plasma and serum were centrifuged at 3000RPM for 10 minutes and separated samples into properly labelledaliquots, stored at -80oC until biochemical analysis was done. Plasma samples used for fasting, postprandial blood sugars and glycated haemoglobin, serum used for lipid profile, uric acid were analysed by laboratory standard methods . hs-CRP was analysed by turbidometric method and IL-6 were determined by enzyme linked immunosorbent assay .The statistical analyses were carried out by using the software Statistical Package for the Social Sciences (SPSS) Version 20.0. The values were expressed as mean ± standard deviation, P < 0.05 was considered statistically significant. Comparisonof study subjects was performed by one-way ANOVA and comparison between the three groups was done by post hoc analysis. Correlation of BMI, Uric acid, hs-CRP and IL-6 with other parameters of the study was done by Pearson’s correlation. The predictive valuesof clinical and descriptive parameters were evaluated by constructing (receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) was calculated.Table 1(see PDF) illustrated that There was a statistically highly significant the mean age of controls (47.27 ± 5.46) whereas it was  for hypertension CAD with and without type 2 diabetes mellitus patientsrespectively (P = 0.0001**). In case of BMI were highly significant different between hypertension CAD with and without type 2 diabetes mellitus patients  When compared to healthy controls .Additionally we observed there was statistically significant of systolic blood pressure  and diastolic blood pressure  in between hypertension CAD with and without type 2 diabetesmellitus patients when compared to healthy controls . The major biochemical and clinical factors associated with CAD diseases were analysed and the results are tabulated in Table 2(see PDF). There weresignificantly elevated levels of FBS, PPBS, HbA1c, total cholesterol, LDL, uric acid, hs-CRP and IL-6 in group 3 and group 2 when compared to group 1, respectively P value is 0.0001**. This inferred that diabetes and hypertension patients were more prone forCoronary artery disease. To further investigation TGL and VLDL concentrations significantly increased in group 2 subjects when compared to group 3 and group 1, The P values were found to be highly statistically significant (P= 0.0001**). We further evaluatedthe influence factors of descriptive, biochemical and clinical parameters compared by Post hoc analysis is represented in Table 3. As described FBS, TC, TGL, HDL, VLDL,LDL, hs-CRP and interleukin-6 were strongly significant, there was moderate significants ofBMI, HbA1c and no significant difference of age, PPBS and uric acid in group 1 vs group 2. When compared with group 1 vs group 3 shown highly significant of age, FBS, PPBS, HbA1c, TC, TGL, HDL, VLDL, LDL, uric acid, hs-CRP, IL-6 and also we observed that BMIwere shown low significant . Additionally we compared group 2 vs group 3 participants shown strongly significants of FBS, PPBS, HbA1c, TC, TGL VLDL, LDL, uric acid, hs-CRP, IL-6, there was a significant of BMI and also no significant difference of age, HDL.To further investigate the relationships among BMI, Uric acid, hs-CRP, interleukin-6, and other indexes were analyzed, and the results were shown in Table 4(see PDF). As described, BMI, uric acid, hs-CRP and interleukin-6 were found to be strongly correlatedto age, SBP, DBP, FBS, PPBS, HbA1c, TC, TGL, LDL, respectively , there were negative correlation between HDL and BMI ,hs-CRP, interleukin-6 . Whereas there is no correlation was observed betweenuric acid and TGL . The diagnostic utility of the uric acid, interleukin-6 and hs-CRP for the detection of early coronary artery disease was compared and results were tabulated in Table 5(see PDF). Among these hs-CRP, showed a stronglysignificant area under curve , there were interleukin -6 has moderate significant . Additionally the uric acid shown statistically significant AUC (0.687), respectively P value is < 0.004*. The present studyshowed that IL-6 have a strong positive correlation with BMI, LDL, uric acid and hs-CRP and therefore increase in serum IL-6 cytokine level led to rise in the levels of other parameters under consideration. The results are shown in Figure 3(see PDF).The current study reveals that circulating plasma levels of IL-6 and CRP and uric acid levels in the studied subjects were associated with a number of clinical cardiovascular risk factors such as age, BMI, SDP, DBP, HbA1c, TC, FBS, PPBS and LDL. Patients withtype-2 diabetes have a twofold increased risk of stroke and all indications of CHD, myocardial infarction (MI), sudden death, and angina pectoris than non-diabetic individuals . InsulinIn our study, serum uric acid levels were found to be substantially higher in patients to cause release of free radicals, which have been linked to inflammatory cell adhesion molecule expression, activation, and adhesion to damaged endothelium result ofthis, which raises the risk of cardiovascular disease . This meData shows that the raised levels of blood pressure and diabetes may be provoke for inflammatory cytokines IL-6, hs-CRP, and uric acid which are promising potential biomarkers for detecting CAD in individuals.