Imaging with positron emission tomography (PET) plays a crucial role in patient selection prior to radioligand therapy and subsequent molecular response assessment. The presented study aims to investigate the role of quantitative uptake parameters on baseline 68Gallium-PSMA-11 PET/CT imaging as to their association with lesion response to radioligand therapy at individual tumor sites. Special emphasis is placed on the utility of PSMA-uptake thresholds for pretherapeutic prediction of lesion response.The prostate-specific membrane antigen (PSMA), a transmembrane protein frequently present on prostate cancer cells, has gained considerable interest as a target for both molecular imaging and therapy. Patients with metastatic castration-resistant prostate cancer can be successfully treated by delivery of beta particle-emitting 177Lu]Lu-PSMA-617. This study aims to quantify lesion-based response to RLT in relation to pretreatment standard molecular imaging metrics derived from [68Ga]Ga-PSMA-11 PET/CT. Sixty-one patients with mCRPC underwent [68Ga]Ga-PSMA-11 PET/CT imaging before and after a median of 4 (IQR 2–6) RLT cycles. Maximum and mean standardized uptake values , as well as tumor-to-liver ratio (TLR), were assessed. A median of 12 (IQR 7–17) lesions was analyzed per patient, resulting in a total of 718 lesions. Lesions with ≥30% SUVmax decline or falling below the blood pool uptake were considered responsive; ≥30% SUVmax increase marked lesion progression. Additionally, 4-point visual scoring was performed according to E-PSMA consensus. In total, 550/718 (76.6%) lesions responded to RLT, including 389/507 (76.7%) bone metastases and 143/181 (79.0%) lymph node metastases. Baseline SUVmax, SUVmean, and TLR values were associated with lesion response by a moderate but significant correlation . For the classification of lesion progression based on baseline PSMA uptake, receiver operating characteristics (ROC) found SUVmax, SUVmean, and TLR to have comparable discriminatory value . Of 42 tumor sites with baseline uptake below the liver (V-score < 2), 19/42 (45.2%) were responsive, 9/42 (21.4%) were stable, and 14/42 (33.3%) showed progression, leaving liver uptake a threshold with low prognostic value for the identification of RLT-refractory lesions (PPV 33%). This was observed accordingly for various liver uptake-based thresholds, including TLR < 1.5, <2.0 with a PPV at 24%, 20%, respectively. Standard uptake parameters quantified by routine baseline [68Ga]Ga-PSMA-11 PET/CT are moderately associated with post-treatment lesion response to [177Lu]Lu-PSMA-617. Commonly applied liver-based uptake thresholds have limited value in predicting refractory lesions at individual tumor sites.Baseline uptake on prostate-specific membrane antigen (PSMA)-targeted imaging is a prerequisite for radioligand therapy (RLT) with [ Inclusion criteria mandated that patients receive [68Gallium was obtained from a 68Ge/68Ga radionuclide generator . [68Ga]Ga-PSMA-11 was administered by intravenous injection and target activity per patient was 1.8–2.5 MBq/kg body weight. Whole-body images (vertex to mid-thigh) were acquired 61 ± 12 min after tracer injection and PET acquisition time was 4 min per bed position. CT data were acquired for attenuation correction and anatomical localization using an X-ray tube voltage of 130 kV with a modulated tube current . Acquisitions were carried out on a Biograph 6 PET/CT scanner , with decay, scatter, and attenuation correction performed in accordance with the procedure guidelines set out by the joint EANM and SNMMI consensus statement Lu-PSMA-617 was administered by slow intravenous injection over 30–60 s, preceded and followed by 1000 mL of saline infusion. RLT was performed as an inpatient procedure at the nuclear medicine therapy ward in accordance with radioprotection regulations. Six cycles with an activity of 7.4 GBq per cycle were intended; administered activities were modified in patients with potential risks for toxicity.Radiolabeling of PSMA-617 with n detail . [177Lu]s). Receiver operating characteristics (ROC) analysis was applied to determine the ability of baseline PET/CT metrics  to predict treatment lesion progression at individual sites. The area under the curve (AUC) was calculated for all lesions and separately for bone metastases and lymph node metastases. Site-specific cutoff values for the detection of PL were calculated using Youden’s J statistic. Various liver-based thresholds were tested as to their discriminatory value for lesion progression. Odds ratios (OR) were calculated with 95% confidence intervals (CI). Statistical analyses were performed with SPSS  and GraphPad Prism . All tests were two-sided, with p-values < 0.05 denominating statistical significance.Results are presented as median with interquartile range (IQR) and mean ± standard deviation (SD) for continuous variables. Categorical variables are reported as frequencies with respective percentages. Comparison of means was performed by a paired t-test for intraindividual analysis or by using a Mann-Whitney U test if data were not normally distributed. Association of categorical parameters was analyzed using non-parametric rank correlation (Spearman’s correlation coefficient denoted with r68Ga]Ga-PSMA-11 PET/CT imaging and subsequently underwent a median of 4 (IQR 3–6) cycles of [177Lu]Lu-PSMA-617 given with a mean treatment activity of 6.9 ± 1.4 GBq per cycle. Cumulative activity per patient was 29.0 ± 17.5 GBq. Of all patients, 31/61 (50.8%) showed ≥50% PSA decline 12 weeks after treatment initiation, while 15/61 (24.6%) showed PSA progression based on PCWG3 criteria (≥25% PSA increase).Overall, 718 lesions were included in the analysis, consisting of 507 bone, 181 lymph node, 22 visceral, and 8 primary/locally recurrent sites in 61 patients with mCRPC (median age 72 [IQR 67–78] years). This corresponded to a median 12 (IQR 7–17) lesions per patient. Patient characteristics at baseline are detailed in max of 14.11 (IQR 8.25–23.01) and SUVmean of 8.72 (IQR 5.09–14.39); tumor-to-liver ratio (TLR) was 3.36 (IQR 1.98–5.70). Of all lesions, 550/718 (76.6%) responded to RLT, consisting of 389/507 (76.7%) bone metastases and 143/181 (79.0%) lymph node metastases. There was no significant difference in mean SUVmax decline in bone vs. lymph node metastases (p = 0.23). Responding lesions (RL) had significantly higher SUVmax, SUVmean, and TLR values at baseline than stable (SL) or progressive lesions (PL), with an SUVmax at 16.01 (RL), 10.82 (SL), 5.11 (PL) (p < 0.001), SUVmean at 9.88 (RL), 6.62 (SL), 3.18 (PL) (p < 0.001), and TLR at 3.89 (RL), 2.55 (SL), 1.36 (PL) (p < 0.001). This was observed in both lymph node and bone metastases ; the relationship is shown in Details on lesion characteristics are provided in tastases , Table 268Ga]Ga-PSMA-11 PET/CT imaging are provided in The course of all lesions per patient is depicted in max, SUVmean, and TLR values. The area under the curve (AUC) was comparable for SUVmax, SUVmean, and TLR with 0.85, 0.87, and 0.83, respectively , with the corresponding SUVmean and TLR thresholds at 4.85  and 1.76 . While liver-based thresholds allowed the stratification of lesions based on their response category in a balanced manner, their value is limited for prognostication, as shown for various liver-derived thresholds in ROC analysis was performed to classify lesion progression based on pretherapeutic SUVectively . Cutoff max decline ≥ 30%) or non-detectability; 28/42 (66.7%) lesions were non-progressive (i.e. SL/RL). Lesions declining to an uptake below the blood pool after RLT (V-score = 0) had lower uptake values at baseline than lesions showing posttherapeutic V-scores ≥ 1 with SUVmax 10.63 vs. 14.89 (p = 0.002), SUVmean 6.78 vs. 9.15 (p = 0.009), and TLR 2.71 vs. 3.48 (p < 0.001) (p = 0.09).In addition to semiquantitative PET measurements, visual scoring (V-score) was noted for all lesions. The V-score change from baseline to posttherapeutic PET assessment was registered, as depicted in < 0.001) B. A mino68Ga]Ga-PSMA-11 PET/CT at baseline are moderately associated with lesion response to [177Lu]Lu-PSMA-617 RLT. Low uptake lesions were more frequently subject to progression, whilst addressable by RLT in a significant fraction of cases. Baseline metrics and liver-based thresholds had only limited value for single-lesion response prediction.This study indicates that routine PET parameters assessed by [177Lu]Lu-PSMA-617 RLT have proven that a higher density of the transmembrane glycoprotein PSMA on prostate cancer cells is associated with increased ligand internalization and treatment efficacy Ga-PSMA-11 PET/CT are moderately associated with lesion response to [177Lu] Lu-PSMA-617. Lesions with uptake values below the liver uptake remain non-progressive in the majority of cases examined in this cohort. Non-dominant tumor sites with low PSMA expression should thus not preclude patients from undergoing RLT.Standard uptake parameters quantified by routine baseline [


Dear Editor,TET2 is one of the most frequently mutated genes in myeloid malignancies . Mor. MorTET2MPN Fig.  6]. Col. ColTET2Nup98-HoxD13 (NHD13) transgenic mouse model, in which ~30% of mice develop AML. Interestingly, TET2 levels were lower in c-kit+ bone marrow (BM) cells of leukemia-transformed NHD13 mice relative to age-matched NHD13 mice, which developed MDS exclusively  or corresponding control (Tet2fl/fl) mice with NHD13 mice and monitored leukemia development following poly(I:C) treatment on both genotypes  counts, splenomegaly and hyper-cellularity in BM, while age-matched NHD13/Tet2-WT mice exhibited only cytopenia . At that time point, neither genotype showed signs of leukemia  population relative to those of Tet2-WT NHD13 mice, whereas the Lin-c-kit+Sca-1+ (LSK) population was unchanged by Tet2 deletion  from pre-leukemic NHD13/Tet2-KO or corresponding control NHD13 mice into lethally-irradiated secondary recipients to assess leukemogenicity. As expected, NHD13/Tet2-KO cell transplantation increased the percentage of CD45.2+ cells and WBCs in peripheral blood (PB) relative to NHD13/Tet2-WT cells  from WT or Tet2-KO mice  pool.To define mechanisms underlying MDS progression, we evaluated the mia Fig. , but Tetice Fig. . Importaon Figs.  and S2E.-KO Fig. . Within ice Fig. . Moreovells Fig.  or that nes Fig. . In the D13 Fig. . Tet2-KOlls Fig. . We nextls Figs. . By 16 w PB Fig. . Notablynts Fig. . Moreoveice Fig.  and obseTet2 loss in HSPCs can lead to hypermutagenicity [+ cells from pre-leukemic NHD13/Tet2-KO vs. matched NHD13/Tet2-WT mice. Relative to NHD13/Tet2-WT mice, we observed 271 newly acquired alterations and 199 alterations with increased variant allele frequency  in NHD13/Tet2-KO mice  and ranked them based on association with AML prognosis (http://precog.stanford.edu)  and mutation ratio (mutation ratio >0.5)  promoted the greatest increase in colony and cell number of NHD13 c-kit+ cells  Table . Accordi.5) Fig. . Pairwisons Fig. . To assesay Fig. . In the lls Fig. . Howeverity Fig. , indicatARIH2 function in leukemogenesis, we retrospectively analyzed GEO datasets and observed lower ARIH2 expression associated with shorter survival in MDS and AML patients  did not, and cells harboring the mutant exhibited a growth advantage relative to ARIH2WT cells  compared to those with TET2-WT  or ascorbate . Relative to vehicle-treated NHD13/Tet2-KO recipients, ASC treatment in NHD13/Tet2-KO mice significantly decreased WBC counts and the frequency of c-kit+ BM cells  from two TET2 mutant high-risk MDS patients (Table + cells (Fig. Given that vitamin C treatment mimics effects of toration , we treals Figs.  and S6A.mia Fig. . Notably KD Fig. . Moreovency Fig. . Finallyts Table  showed tlls Fig. .In summary, our results indicate that TET2 activity prevents further transformation of MDS HSPCs by decreasing the occurrence of secondary mutations, and that pharmacological enhancement of TET activity may represent an optimal strategy to block MDS malignant transformation.Supplementary data


Continuous emergence of the Omicron variant, along with its subvariants, has caused an increasing number of infections, reinfections, and vaccine-breakthrough infections, seriously threatening human health. Recently, several new Omicron subvariants, such as BA.5, BA.2.75, BA.4.6, and BF.7, bearing distinct mutation profiles in their spike (S) proteins, have significantly increased their capacity to evade vaccine-induced immunity and have shown enhanced infectivity and transmissibility, quickly becoming dominant sublineages. In this study, we found the S proteins of these Omicron subvariants to have 2- to 4-fold more efficient membrane fusion kinetics than that of the original Omicron variant (BA.1), indicating that these novel Omicron subvariants might possess increased pathogenicity. We also identified that peptide-based pan-CoV fusion inhibitors, EK1 and EK1C4, showed equal efficacy against membrane fusion mediated by S proteins of the noted Omicron subvariants and infection by their pseudoviruses. Additionally, either immune sera induced by wild-type (WT) SARS-CoV-2 RBD-based vaccine or BA.2 convalescent sera showed potent synergism with EK1 against both WT SARS-CoV-2 and various Omicron subvariants, further suggesting that EK1-based fusion inhibitors are promising candidates for development as clinical antiviral agents against the currently circulating Omicron subvariants. In late 2021, the Omicron variant B.1.1.529/BA.1 was first identified in South Africa and quickly spread to many countries ,4. SubseDifferent Omicron subvariants contain distinct mutation profiles, especially in their S proteins, well known to play a crucial role in mediating viral infection. Compared with WT SARS-CoV-2 S protein, Omicron BA.1 contains more than 30 spike mutations. In particular, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H mutations are known to occur in the Omicron BA.1 receptor binding domain (RBD) . Such mutations significantly alter the conformation of RBD in S protein and, hence, seriously threaten clinical vaccine efficacy . BA.2.12in vivo [Previous studies reported that S-mediated fusogenicity plays an important role in viral pathogenesis . For exain vivo . On the in vivo . NeverthAfter receptor engagement, heptad repeat 1 (HR1) and 2 (HR2) regions in the S2 subunit of coronavirus S protein interact to form a six-helix bundle (6-HB) structure, which, in turn, drives viral fusion with and entry into the host cell . Our preIn the current study, we found that the recently emerging Omicron subvariants showed strengthened fusion kinetics, particularly BA.2.75, BA.4.6, BA.5, and BF.7, compared to that of BA.1, indicating that they possess increased pathogenicity. Although these circulating Omicron subvariants carry distinct mutation profiles in S protein, their HR1 functional domains remain conserved. Accordingly, we found that our EK1-based fusion inhibitors  maintainWe first evaluated the fusogenicity of different Omicron subvariants. To mimic authentic viral fusion with target cells, as mediated by S protein, their S proteins were expressed on the membrane surface of 293T-GFP cells, as effector cells (293T-S-GFP)  of 142.02, 172.45 and 65.84 nM, respectively, while EK1C4 showed even more efficacy with IC50s ranging from 3.67 nM to 5.07 nM . On BA.21/2: 24h) and potential oral bioavailability by targeting SARS-CoV-2 HR1 in S2 subunit and RBD in S1 subunit [To further assess the efficacy of EK1-based pan-CoV fusion inhibitors against Omicron subvariants, we developed lentivirus-based nonreplicative pseudovirus systems for these Omicron subvariants. Such constructs can effectively mimic the entry process of authentic virus and are, therefore, widely used to evaluate antiviral agents . Both EK subunit . At the  subunit . These f subunit  and IPB2 subunit , respectCurrently, more than 12 billion vaccine doses have been administered globally , includiSince both RBD and HR1 are key in mediating viral entry , it is rMore importantly, WT-RBD-immunized mouse sera showed weak inhibitory activity against BA.2.12.1 and BA.2.75 with 31.4% and 33.1% inhibition, respectively, as did EK1 (100 nM) with 33.2% and 30.6% inhibition, respectively. However, when combined, they showed increased efficacy to 62.3% inhibition on BA.2.12.1 and 63% inhibition against BA.2.75  alone showed moderate inhibitory activity against the BA.2.12.1 sublineage with 64.6% inhibition, but little efficacy against BA.2.75, BA.4/BA.5, BA.4.6, BF.7, and WT SARS-CoV-2 . However, BA2-convalescent sera, when combined with low-dose EK1 (100 nM), showed significantly improved efficacy against BA.2.12.1, BA.2.75, BA.4/BA.5, BA.4.6, and BF.7 in the range of 57.9% to 83.0% inhibition ; HeLa and Caco2 cell lines were from the Chinese Academy of Science Cell Bank . 293T/ACE2 cells were preserved in our laboratory. All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). Plasmids, including pAAV-SARS-CoV-2-S-D614G-IRES-EGFP, pAAV-SARS-CoV-2-S-Delta-IRES-EGFP, pAAV-SARS-CoV-2-S-Omicron-IRES-EGFP, and PC-hACE2/horse_ACE2/cattle_ACE2/swine_ACE2/rabbit_ACE2/civet_ACE2/ bat_ACE2, were synthesized or preserved in our laboratory. Peripheral blood samples were collected from a convalescent BA.2 patient. Serum sample was isolated from centrifuged blood sample for inhibiting pseudovirus infection with 1:500 dilution. All collections were conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Ethics Committee of Shanghai Fourth People's Hospital (2022095-001).Briefly, WT SARS-CoV-2 RBD-Fc (5 µg) formulated with an equal volume of Imject Alum adjuvant (Thermo Scientific) was used to vaccinate Balb/c mice (six-week-old) three times at two-week intervals, as previously described ,35. At tWestern blot was performed using an anti-SARS-CoV-2 S antibody and an anti-actin antibody, as previously described . BrieflyPlasmid pAAV-IRES-S-EGFP, encoding S protein and EGFP, was transfected into 293T effector cells (293T/S/GFP). Caco2 cells, naturally expressing human ACE2 receptors on the membrane surface, were used as target cells. 293T cells, transfected with plasmid pAAV-IRES-EGFP (293T/EGFP), were used as negative control. Effector cells (293T/S/GFP) were collected and resuspended. Free effector cells were added into target cells  for coincubation for indicated time at 37 °C and then observation under the fluorescence microscope.HIV-1 backbone-based Pseudovirus (PsV) bearing wild or mutant SARS-CoV2 S protein was produced, as previously described . Caco2 ct-test and ANOVA test. P values less than 0.05 were significant; **P < 0.01; ***P < 0.001. The concentration for half inhibition (IC50) was calculated by CalcuSyn software [Statistical analyses were carried out using GraphPad Prism 8.0. Analyses of independent data were carried out through Student’s unpaired two-tailed software .Click here for additional data file.


In 2019, the World Health Organization (WHO) endorsed thermal ablation (TA) for use within “screen-and-treat” cervical cancer prevention programs in low- and middle-income countries (LMICs), including among women living with HIV (WLWH). We evaluated TA efficacy for treatment of biopsy-confirmed cervical intraepithelial neoplasia grades 2 and 3 (CIN2/3) among WLWH in western Kenya .Between August 2019 and November 2020, WLWH age 25-65 years underwent high-risk human papillomavirus (hrHPV) self-collection. hrHPV-positive women underwent colposcopy-directed biopsies, and thermal ablation treatment if eligible per WHO guidelines. Women with biopsy-confirmed CIN2/3 had colposcopy-directed biopsies at 12-months to determine treatment efficacy.Sixty-eight hrHPV-positive WLWH with biopsy-confirmed CIN2/3 at baseline; 14 CIN2, 54 CIN3, underwent thermal ablation. Mean age and parity were 41.2 years and 4, respectively. The mean CD4 count was 473.98 cells/mm3 and 96.9% had HIV viral suppression. Fifty-eight women (83.8%) have been seen for a 12-month follow-up visit, and pathology results are available for 54 (79.4%). Of these, 35 (66.0%) had successful treatment, defined as biopsy-confirmed CIN1 or normal findings 12-months following treatment, while 18 (34.0%) had treatment failure - persistent biopsy-confirmed CIN2/3. Treatment failure was 23.1% 95% CI (13.0 to 45.9) and 37.5%, 95% CI (22.1 to 52.0) among women with CIN2 and CIN3 at baseline, respectively.Hand-held thermal ablation devices are affordable, portable, easy to use, and hence highly scaleable within screen-and-treat programs in LMICs. However, our preliminary results, with rigorous disease status verification at both baseline and follow-up find higher than previously reported treatment failure rates for CIN3 among WLWH, a high-risk population for cervical cancer. If replicated by larger studies, this highlights a potential limitation of the current WHO cervical cancer elimination strategy, calling for better risk stratification in this population, and/or consideration of adjuvant therapy to prevent CIN2/3 recurrence following thermal ablation.


Dysregulation of epigenetic mechanisms have been depicted in several pathological consequence such as cancer. Different modes of epigenetic regulation (DNA methylation (hypomethylation or hypermethylation of promotor), histone modifications, abnormal expression of microRNAs (miRNAs), long non-coding RNAs, and small nucleolar RNAs), are discovered. Particularly, lncRNAs are known to exert pivot roles in different types of cancer including breast cancer. LncRNAs with oncogenic and tumour suppressive potential are reported. Differentially expressed lncRNAs contribute a remarkable role in the development of primary and acquired resistance for radiotherapy, endocrine therapy, immunotherapy, and targeted therapy. A wide range of molecular subtype specific lncRNAs have been assessed in breast cancer research. A number of studies have also shown that lncRNAs may be clinically used as non-invasive diagnostic biomarkers for early detection of breast cancer. Such molecular biomarkers have also been found in cancer stem cells of breast tumours. The objectives of the present review are to summarize the important roles of oncogenic and tumour suppressive lncRNAs for the early diagnosis of breast cancer, metastatic potential, and chemotherapy resistance across the molecular subtypes. Epigenetic dysregulations have a crucial impact on the development and progression of human cancers, including breast cancer . Epigenep = 0.003) in malignant samples could separate it breast cancer samples from normal control  and correlated with advanced TNM stage (p = 0.002), poorer pathological differentiation (p = 0.004), and shorter overall survival (SOS) and disease-free survival (DFS) (p < 0.0001) in progesterone receptor (PR)  positive cancer tissues (p < 0.00001). Moreover, validation using gene expression omnibus data sets  and 100 breast cancer patients confirmed similar results  . A meta- tissues . Moreove results . Based o results . Higher  results . Additio results . Chen et results . Further results .via the regulation of SNCG (Synuclein Gamma) expression  during cancer progression. LncRNA HOXD-AS1 interacts with miR-421 and inhibits its expression leading to the upregulation of SOX4, a master regulator of EMT . Similarpression . High expression . Zheng epression . In addipression . Also, hpression . Si et apression . Further-5p axis .via increasing Checkpoint kinase 2 (CHK2) phosphorylation , PARP (cleaved-Caspase-3 and cleaved-poly adenosine diphosphate-ribose polymerase) had elevated expression in low expression lncRNA BANCR group . Liu et  pathway . Elevate pathway . Further pathway . The exp pathway .It was observed that lncRNAs can also regulate several cancers associated signalling pathways, including the activation of transcription factors, such as nuclear factor kappa B (NF-κB). For example, overexpressed lncRNA NKILA bound to NF-κB/IĸB masked its phosphorylation. This interaction prevented the over-activation of the NF-κB pathway in inflammation stimulated breast epithelial cells . Accordivia modulation of miR-200b/axis/Wnt/β-catenin pathway  signalling pathway . Dysreguviz., lncRNA YIYA regulates CDK6 (cell division protein kinase 6) dependent phosphorylation of PFKFB3 (fructose bis-phosphatase PFK2), and thus can convert glucose 6-phosphate (G6P) to fructose-2,6-phosphate  gene in breast cancer (via modulating the expression of miR-4766-5p and SIRT1 (Sirtuin 1) genes (in situ hybridization) and Western blot assays in MCF-7 and MDA-MB-231 breast cancer cell. Luciferase reporter assay validated that RHPN1-AS1 inhibits miR-4261 and regulates the direct transcriptional target of c-Myc (Immunoprecipitation assays provided evidence that lncRNA H19 regulated the expression of t cancer . The res1) genes  Higher eof c-Myc .p = 0.0086)  . Huang a 0.0086) . Another systems . Accordi systems .Oncogenic lncRNA LINC02163 was found to be involved in breast cancer pathogenesis by mode of LINC02163/miR-511-3p/HMGA2 (high mobility group A proteins 2) axis . The finLi and et al. found that upregulation of lncRNA ZFHX4-AS1 suppresses FAT4 and increases YAP1 (yes-associated protein 1) and TAZ (Tafazzin) gene expression which is attributed to breast cancer cell proliferation . A reporp = 0.022), lymph node metastasis (p = 0.020), and higher Ki-67 positivity (p = 0.017). A multivariate analysis suggested that a low level of lncRNA EGOT acts as an independent prognostic factor for poor survival rate in breast cancer patients  (There are a number of lncRNA whose downregulation contributes in breast cancer development and progression . A meta-= 0.039) . Further= 0.039) . Yang et= 0.039) . Low relvia modulating expression of cyclin A2, CDK2, p-Akt, p-p44/42 MAPK, and p-p38 MAPK cancer signalling molecules (in-vivo and in-vitro model system induced the EMT process in cancer via regulation of PI3K (phosphatidylinositide-3 kinase)/Akt pathways. Therefore, MALAT1 may act as a promising therapeutic target for breast cancer metastasis via the PI3K-Akt pathway  expression. Thus, the results suggested that lncRNA MAGI2-AS3 has the potential to serve as an anticancer therapeutic candidate . Tumour olecules . Similarolecules . LncRNA olecules . Downreg pathway .A study by via the miR-190b-5p/MYLIP (Myosin regulatory light chain interacting protein) axis, thus providing evidence for potential therapeutic targets for breast cancer patients  and its binding with co-activator YAP1, leading to reduced metastatic ability . Anotherpatients . On the patients .in situ (DCIS) breast cells  identified lncRNA LINC00885 expression in both normal and ductal carcinoma st cells . Expressst cells . Overexpst cells . Similarst cells . Furtherst cells . In addist cells .Gene expression profiling deciphered the breast cancer into four distinct molecular subtypes such as Luminal, Her2+, Her2 enriched, TNBC, and basal like. Patients with same molecular subtype responded differently to targeted therapy and showed diverse clinical outcomes. However, the exact underlying mechanism for molecular heterogeneity remains to be elucidated. Many researchers have evaluated molecular subtype specific lncRNAs expression in breast cancers and suggested its involvement in cancer molecular heterogeneity . Computain-vivo and in-vitro systems  gene expression restoration . Further systems . Another systems . Greater systems . Presenco ELAVL1 . Beltráno ELAVL1 . Higher o ELAVL1 . By asseo ELAVL1 . Another pathway . Wang et pathway . RNA imm pathway . Further pathway .via targeting miR-205 , and ALDH1 (Aldehyde dehydrogenase 1) marker’s expression . Similar miR-205 . Microar miR-205 . LncRNAs miR-205 .Chemotherapy resistance is the major cause of cancer related deaths. LncRNAs have a key role in developing resistance against radiotherapy, chemotherapy, immunotherapy, and targeted therapy. Inhibition of lncRNA LINC02582 expression increased radiosensitivity miR-200c/LINC02582/CHK1 in breast cancer samples . LncRNA via the H19/SAHH/DNMT3ß (DNA (cytosine-5)-methyltransferase 3 beta) axis, contributed to tamoxifen resistance in breast cancer  . BermejoLong ncRNAs like H19 and XIST were discovered in the pre-genomic era, but were not fully characterized and explored until the early 2000s . Inventi


Sotrovimab, a monoclonal antibody with efficacy against SARS-CoV-2 including certain Omicron variants, has been used in treatment of mild-moderate COVID-19. Limited data exists regarding its use in pregnant women.Electronic medical record review of pregnant COVID-19 patients treated with sotrovimab from 12/30/21-1/31/22  was performed. Included were pregnant individuals ≥ 12 years, weighing ≥ 40 kg, with positive SARS-CoV-2 test (within 10 days). Those receiving care outside YNHHS or receiving other SARS-CoV-2 treatment were excluded. We assessed demographics, medical history, and Monoclonal Antibody Screening Score (MASS). Clinical outcomes assessed included emergency department (ED) visit < 24 hours, hospitalization, ICU admission, and/or death within 29 days of sotrovimab. Pregnancy and neonatal outcomes were assessed until 8/15/22.2. Sixty-three percent were Caucasian, 9% Hispanic, 14% African-American, and 9% Asian. Nine percent had diabetes and sickle cell disease. Five percent had well-controlled HIV. Eighteen percent, 46%, and 36% received sotrovimab in trimester 1, 2, and 3, respectively. No infusion/allergic reactions occurred. MASS values were < 4. Only 12/22 (55%) received complete primary vaccination ; none received a booster.Among 22 subjects, median age was 32 years and body mass index was 27 kg/mThere were no ICU admissions nor deaths. One subject was hospitalized for post-partum pyelonephritis; another had an ED visit for post-partum vaginal bleeding. Median gestational age at birth was 38.9 weeks. Nine percent had premature labor and premature rupture of membranes, respectively. Median infant birth weight was 3220 g. One neonate required an ICU stay due to prenatally diagnosed omphalocele (before sotrovimab) in a mother with congenital defect history. There were no abortions, fetal loss, or other birth/neurodevelopmental defects.Pregnant COVID-19 patients receiving sotrovimab at our center tolerated it well with good clinical outcomes. Pregnancy and neonatal complications did not appear sotrovimab-related. Though a limited sample, our data helps elucidate the safety and tolerability of sotrovimab in pregnant women.All Authors: No reported disclosures.


Waning protection against emerging SARS-CoV-2 variants by pre-existing antibodies elicited because of current vaccination or natural infection is a global concern. Whether this is due to the waning of immunity to SARS-COV-2 remains unclear.We aimed to investigate the dynamics of antibody isotype responses amongst vaccinated naïve (VN) and naturally infected (NI) individuals.n = 100) in two phases: phase-I (P-I) at ~ 1.4 and phase-II (P-II) at ~ 5.3 months. Antibody levels were compared with those of unvaccinated and naturally infected subjects  at ~ 1.7 (P-1) and 5.2 (P-II) months post-infection. Neutralizing antibodies (NTAb), anti-S-RBD-IgG, -IgM and anti-S-IgA isotypes were measured.We followed up antibody levels in COVID-19 messenger RNA (mRNA)-vaccinated subjects without prior infection  than the NI group at P-I, except for IgM. In the VN group, a significant waning in antibody response was observed in all isotypes. There was about an ~ 4-fold decline in NTAb levels (P < 0.001), anti-S-RBD-IgG , anti-S-RBD-IgM  and anti-S1-IgA . In the NI group, a significant but less steady decline was notable in S-RBD-IgM , and a much smaller but significant difference in NTAb  anti-S-RBD IgG . Unlike the VN group, the NI group mounted a lasting anti-S1-IgA response with no significant decline. Anti-S1-IgA, which were ~ 3-fold higher in VN subjects compared with NI in P-1 (P < 0.001), dropped to almost the same levels, with no significant difference observed between the two groups in P-II.The VN group elicited significantly greater antibody responses (Whereas double-dose mRNA vaccination boosted antibody levels, vaccinated individuals’ ‘boost’ was relatively short-lived. The recent Coronavirus disease 19 (COVID-19) outbreak caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has put the globe in an emergency state.,On 21 December 2020, Qatar initiated a mass COVID-19 vaccination programme, first utilizing the BNT162b2 vaccine and then the mRNA-1273 (Moderna) vaccine.,,When vaccination was ramped up, the country had two back-to-back waves from January to June 2021, mainly dominated by the B.1.1.7  and B.1.351 (or beta) variants.According to current evidence, patients vaccinated against COVID-19 would lose around half of their neutralizing antibodies (NTAb) ~ 108 days post-vaccination.Although vaccine-induced immunity is being extensively studied, the body of evidence for infection-induced immunity is very limited and insufficient to establish an antibody titre threshold that shows whether a person is protected from infection.n = 100) who had received two doses of the two approved mRNA vaccines in Qatar: BNT162b2 and mRNA-1273. Levels were compared with those of unvaccinated but NI subjects (n = 40). Venous blood samples were collected from vaccines in two phases: phase 1 (P-I) and phase (P-II). For the VN group, P-I samples were collected at 1.4 months (median = 6 weeks) and P-II samples were collected at 5.3 months (median = 23 weeks) after the second dose. For the NI group, P-I samples were collected at ~ 1.7 months (median = 7 weeks) and P-II samples were collected at 5.2 months (median = 22 weeks) post-infection with SARS-CoV-2.The study was reviewed and approved by the Institutional Review Board at Qatar University (QU-IRB 1537-FBA/21). The study included VN participants , i ≥ 1.00 Positive (IgM antibodies to SARS-CoV-2 detected). Architect automated chemiluminescent assay  was used to test the samples for the previous infection by measuring the SARS-CoV-2 anti-nucleoprotein IgG antibodies (anti-N), considering that the IgG antibodies produced against the RBD on the spike protein are different from the IgG antibodies produced against the nucleoprotein of the virus. Therefore, the positive anti-N results of SARS-CoV-2 anti-nucleoprotein IgG antibodies indicate previous exposure to the whole virus.After blood collection, plasma was separated by centrifugation and heat-inactivated. Serological testings were performed using the automated analyser CL-900i® from Mindray Biomedical Electronics 20-22 to detect: (1) NTAb  were measured using the automated analyser CL-900i®. This assay is a competitive binding chemiluminescent immunoassay for the quantitative determination of SARS-CoV-2 NTAb that blocks the interaction between the receptor binding domain (RBD) of viral spike protein (bound on magnetic beads) and the enzyme-conjugated ACE2 surface receptor. Phosphate-buffered saline (PBS) was used to dilute the samples with readings higher than the mentioned range. The assay has a WHO conversion factor of 1 AU = 3.31 IU/mL, and the reference range is 10 AU/mL to 400 AU/ml. We recently evaluated this novel assay and reported high specificity and sensitivity against two reference methods.P-values were two-sided at a significance level of 0.05.GraphPad Prism software  was used to perform the statistical analysis. Continuous variables were summarized using geometric means and 95% confidence intervals (95% CIs). The collected dataset was subjected to the Shapiro–Wilk normality test to evaluate the data’s normality. Because of the absence of normal distribution, nonparametric tests were performed using Wilcoxon rank-sum test for pairwise group comparisons and Mann–Whitney U to test for the differences between independent samples. In the different scatter plots, the central horizontal bar line shows the geometric mean titre, and the error bars show the 95% CIs. All n = 100) had no previous history of infection  and received two doses of either BNT16b2 or mRNA-1273. After receiving the second dose in P-I and P-II, the median weeks were 6 (1.4 months) and 23 (5.3 months) weeks, respectively (n = 40) were unvaccinated COVID-19-recovered individuals. In P-I 1 and P-II, the median weeks were 7 (~ 1.7 months) and 22 (5.2 months) weeks post-COVID-19 infection, respectively. The NI group comprised 20% females and 80% males  and 26% (12/47), respectively. In P-II, 100% remained positive for anti-S-RBD IgG and 99% (99/100) were positive for NTAb antibodies. The anti-S1-IgA positivity rate dropped to 92% (86/93), and only 2% (1/47) remained positive for the IgM.Amongst the NI participants in P-1, the positivity rate for NTAb antibodies was 90% (35/39). For anti-S-RBD-IgG, IgM and IgA, the positivity rates were 100 (31/31), 43 (17/40) and 72% (23/32), respectively. In P-II, 90% (35/39) were positive for NTAb antibodies. However, the positivity rates dropped to 97 (30/31), 13 (5/39) and 66 (21/32) for anti-S-RBD-IgG, -IgM and -IgA, respectively.P < 0.001) in the levels of NTAb antibodies was observed, from the geometric mean 1328.4 (95% CI: 1086.5–1624.2) to 317.94 IU/mL (95% CI: 261.5–386.5 IU/mL) (P < 0.001) was observed in the levels of anti-S-RBD-IgG antibody levels from the geometric mean 1950.93 (95% CI: 1617.5–2353.1) to 366.107 BAU/mL (95% CI: 294.4–455.20) (P < 0.001) from the geometric mean 0.56 (95% CI: 0.4–0.8) to 0.09 (95% CI: 0.04–0.25) (P < 0.0001) from the geometric mean 6.30 (95% CI: 5.4–7.3) to 3.19 (95% CI: 2.6–3.9)  . A signi–455.20) . IgM dec04–0.25) . The lev2.6–3.9) .P < 0.001), from the geometric mean 157.3 IU/mL, 95% CI: 108.7–227.7 to 107.0 IU/mL, 95% CI: 80.42–142.3 (P < 0.001) from the geometric mean 66.54 (95% CI: 36.81–120.3) to 58.65 BAU/mL (95% CI: 37.06–92.80) (p = 0.005) (P < 0.001)  . IgM dec< 0.001) . No sign< 0.001) .,To the best of our knowledge, this is the first study to comprehensively evaluate the levels of SARS-CoV-2 neutralizing, anti-S-RBD-IgG, Anti-S-RBD-IgM and anti-S1-IgA antibodies in VN and unvaccinated NI individuals. In the current study, mRNA vaccination elicited significantly greater NTAb, anti-S-RBD- IgG and anti-S1-IgA, compared with natural immunity . These r,,However, despite the mRNA vaccination-boosted antibody levels, this ‘boost’ was relatively short-lived, with ~ 2- to 6-fold significant waning observed in NTAb, anti-S-RBD-IgG, anti-S-RBD-IgM and anti-S1-IgA, 23 weeks post-full-vaccination . These rIn comparison with mRNA vaccine-induced immunity, natural infection elicited a significant but less steady ~ 1–2-fold decline in NTAb, anti-S-RBD-IgG, and anti-S-RBD-IgM . InteresThese findings are similar to a recent report showing that natural infection exhibit a lasting IgA response.,,,This study had several limitations. It is known that recent variants, such as Omicron, need many folds of antibody titre to be neutralized compared to the original strains. That is, the mutations that emerged in different variants of the SARS-CoV-2 genome should have a significant influence on viral protein structures, shape, function and immunogenicity, which, in theory, should greatly affect the strength and the effectiveness of the immunological response and possibly duration of antibody’s waning in infected patients. It would be great to study the antibody response and waning against different variants. However, it was not applicable to include variant-specific data because of the limited number of samples and lack of sequencing data. In addition, a variety of variables could influence the level of immune response elicited after infection. It should be noted that our NI group included only 45% symptomatic subjects, whereas the remaining were paucisymptomatic (20%), asymptomatic (20%) or with unspecified severity (15%), which could have affected our results. In those with more severe COVID-19, NTAb antibody titers were reported to rise faster and reach a greater peak.,Furthermore, several studies have shown a link between cycle threshold (Ct) and antibody titre, with lower Ct values linked with greater antibody titers at the population level.Despite these limitations, this study has several strengths that merit attention. First, most of the published studies have mainly focused on NTAb, IgG or IgM, whereas studies on IgA response are minimal, particularly amongst unvaccinated NI subjects. Second, in this study, we assessed anti-N antibodies, which is crucial to identify those who were exposed to a virus but were asymptomatic prior to vaccination, especially amongst those vaccinated with vaccines containing only S protein. In addition, despite the relatively small sample size across the analysed groups, we utilized strict inclusion criteria and included participants from a wide age range to achieve valid comparisons.Our findings provide important insights into the durability of vaccine- and natural infection-induced immunity. We evaluated the antibody responses of NTAb, anti-SRBD IgM, anti-S1-IgA and anti-SRBD IgG antibodies. Whereas double-dose mRNA vaccination elicited higher antibody titers compared with natural infection, this ‘boost’ was relatively short-lived in vaccinated individuals. In contrast, natural infection exhibited a less steady decline in NTAb antibodies, IgG, IgM and a lasting IgA response. Understanding the degree of waning immunity is crucial for policymaking, particularly regarding vaccination strategies, supporting the consideration of booster doses to sustain protection against COVID-19.Conceptualization, H.A.S., H.M.Y., L.J.A.-R. and G.K.N.; methodology, H.A.S., B.A.H., S.Y. and F.M.S., software, B.A.H., S.Y. and F.M.S; validation, H.A.S., B.A.H., S.Y. and F.M.S.; formal analysis, H.A.S., G.K.N., B.A.H. and S.Y.; Investigation, H.A.S., A.I., H.M.Y., L.J.A.-R. and G.K.N.; resources, H.A.S., N.L., H.Q., N.A.D., H.M.Y., L.J.A.-R and G.K.N.; data curation, H.A.S., B.A.H., S.Y. and G.K.N.; writing—original draft preparation, H.A.S., S.Y. and G.K.N.; writing—review and editing, H.A.S., S.Y., N.Y., D.W.A., N.A.D., H.M.Y., L.J.A.-R and G.K.N; visualization, H.A.S., B.A.H., S.Y., N.Y., D.W.A., H.M.Y., L.J.A.-R A.I. and G.K.N; supervision, H.A.-S., H.M.Y. and G.K.N.; project administration, H.A.S., H.M.Y., L.J.A.-R and G.K.N.; funding, G.K.N. All authors have read and agreed to the published version of the manuscript.


Streoptomyces rimosus M527 is a producer of the polyene macrolide rimocidin which shows activity against various plant pathogenic fungi. Notably, the regulatory mechanisms underlying rimocidin biosynthesis are yet to be elucidated.rimR2, which located in the rimocidin biosynthetic gene cluster, was first found and identified as a larger ATP-binding regulators of the LuxR family (LAL) subfamily regulator. The rimR2 deletion and complementation assays were conducted to explore its role. Mutant M527-ΔrimR2 lost its ability to produce rimocidin. Complementation of M527-ΔrimR2 restored rimocidin production. The five recombinant strains, M527-ER, M527-KR, M527-21R, M527-57R, and M527-NR, were constructed by overexpressing rimR2 gene using the promoters permE*, kasOp*, SPL21, SPL57, and its native promoter, respectively, to improve rimocidin production. M527-KR, M527-NR, and M527-ER exhibited 81.8%, 68.1%, and 54.5% more rimocidin production, respectively, than the wild-type (WT) strain, while recombinant strains M527-21R and M527-57R exhibited no obvious differences in rimocidin production compared with the WT strain. RT-PCR assays revealed that the transcriptional levels of the rim genes were consistent with the changes in rimocidin production in the recombinant strains. Using electrophoretic mobility shift assays, we confirmed that RimR2 can bind to the promoter regions of rimA and rimC.In this study, using domain structure and amino acid alignment and phylogenetic tree construction, rim genes and binding to the promoter regions of rimA and rimC.A LAL regulator RimR2 was identified as a positive specific-pathway regulator of rimocidin biosynthesis in M527. RimR2 regulates the rimocidin biosynthesis by influencing the transcriptional levels of The online version contains supplementary material available at 10.1186/s12934-023-02039-9. Streptomyces, a genus of Gram-positive bacteria with three types of PKSs , is best known for producing polyketides [Fusarium oxysporum f. sp. cucumerinum [Polyketides, a large group of secondary metabolites synthesized by polyketide synthases (PKSs), exhibit various bioactivities, including antifungal (rimocidin), antibacterial (penicillin), antitumor (daunorubicin) properties –3. They yketides –6. Polyeyketides –9, They yketides , nystatiyketides , amphoteyketides , and rimyketides . For exaumerinum , is a prStreptomyces causes bottlenecks, leading to low production levels and long fermentation periods [Streptomyces is a complex process involving multiple levels [However, polyketide biosynthesis in  periods . Secondae levels –18, incle levels –21.Streptomyces antibiotic regulatory protein (SARP) family regulator, such as ActII-orf4, which regulates actinorhodin biosynthesis, and CcaR, which regulates clavulanic biosynthesis. These regulators are characterized by the presence of OmpR -like DNA-binding domains [Streptomyces natalensis [Streptomyces nodosus [Streptomyces noursei [Streptomyces ahygroscopicus [S. noursei ATCC 11,455 [S. natalensis [Streptmyces avermilitis [To date, the different types of regulators involved in polyene macrolide biosynthesis have been categorized as follows: (1)  domains . (2) PAS domains , 24. Thetalensis . AmphRIV nodosus , and Nys noursei . (3) Lar noursei  having ascopicus , and NysC 11,455 . (4) SARtalensis , and Ptermilitis . Recent rmilitis –35.Streptomyces rimosus M527, a rimocidin producer, was originally isolated by Lu et al. [S. rimosus M527. However, to the best of our knowledge, no pathway-specific rimocidin biosynthesis regulators are currently known.u et al.  and depou et al. , fermentu et al. , were apStreptomyces diastaticus var. 108 has been predicted, and its biosynthetic gene cluster has been published (GenBank Accession No. AY442225) [S. rimosus M527 was sequenced (GenBank Accession No: NZ_SADA00000000.1), a biosynthetic gene cluster responsible for rimocidin production (GenBank Accession No. MK300953) , rimR2-deleted and rimR2-complemented strains. Subsequently, rimR2 gene was overexpressed using different promoters  to improve rimocidin production. Furthermore, the regulatory mechanism of RimR2 was identified using electrophoretic mobility shift assays (EMSA).Although the rimocidin biosynthetic pathway in Y442225) , no pathS. rimosus M527 genome sequence, rimR2 gene (2757 nucleotides (nt)), located in the rimocidin biosynthesis gene cluster, encodes a protein with a predicted molecular mass of 97.3 kDa consisting of 918 aa. RimR2 protein contains a conserved nucleotide phosphate-binding domain  and an HTH DNA-binding domain   method  analysis revealed that the mutant M527-ΔrimR2 could not produce any rimocidin, whereas a distinct rimocidin peak was clearly observed in the WT culture filtrates  located in the gene cluster. The mutant M527-ΔrimR2 exhibited significantly lesser transcriptional levels of all the candidate rim genes than the WT strain  was performed to examine the effects of ain Fig. , and therim genes. In this experiment, His6-tagged RimR2 protein was generated in E. coli BL21 (DE3) , showing an 81.8% increase compared with the WT strain (207.2 mg/l) , M527-R3, and M527-R4 were determined via a shake-flask experiment  is the consensus nucleotide sequence of the binding site of PAS-LuxR regulators has been revealed [S. rimosus M527 and found three matches similar to the sixteen conserved nucleotide sequences: (1) CTAGGGAATTCCCGAG, which was the most similar to the consensus sequence. It is located 103-bp upstream of open reading frame 18, which encodes putative GDP-mannose 4,6-dehydratase, but does not lie within its promoter region; (2) GCCAGGAATTCCCGCA, situated near the 3′-end of the internal sequence of rimF, which encodes an aminotransferase, but does not lie within the putative promoter region of rimG encoding cytochrome P450 monooxygenase; (3) ACCGGAAAATCCTTAG, which is present in the intergenic region of rimE and rimD, 100-bp upstream of rimE but not within its putative promoter region. The locations of these three sequences suggest that they do not comprise the core elements for gene expression, which may explain the limited regulatory effect exerted by RimR1 on rimocidin production. The mechanism whereby RimR1 regulates structural genes in the rimocidin gene cluster will be elucidated in a future study.RimR1 shares high similarity with several well-studied transcriptional regulators of the PAS-LuxR family, for example, it shares 49.61% amino acid sequence identity with PimM from talensis . Notablytalensis . Surprisrevealed –47. UsinrimR1 promoter. Moreover, qRT-PCR revealed that rimR2 deletion decreased rimR1 expression and restored it to a level comparable with that in the WT strain M527 when it was complemented, suggesting that RimR2 indirectly regulates rimR1 expression. The relationship between RimR1 and RimR2 and their regulatory hierarchy are also worth investigating in a future study.The biosynthetic gene clusters encoding polyene macrolide antibiotics have been sequenced and multiple regulatory genes, usually organized in a hierarchical network, have been identified within them , 31, 42.S. rimosus M527. These two tetraenes differ in the aglycone moiety, with a propyl group in rimocidin and a methyl group in CE-108. As the elongation module is common for both rimocidin and CE-108 biosyntheses, RimR2 regulates both biosyntheses almost identically  and YrimR1R (5′-GATGAAGCCCTCGACGACAC-3′) were designed following the rimR1 gene sequence (GenBank accession no. MK300953).RNA extraction and the analysis of the transcriptional level of o et al. . qRT-PCRrimR2 coding sequence was amplified by PCR using primers PrimR2-F2/R3 . RimR2 protein was purified using nickel-NTA column (Qiagen) and eluted using imidazole. The inducible expression and purification of were performed according to standard manipulation method described by Sambrook and Russel [A 2757-bp DNA fragment harboring the rim genes were amplified by PCR using the biotin labeled primers  for the identification of clusters involved in rimocidin, and function annotation of biosynthetic gene cluster was listed in t test was used for statistical analysis.All experiments were carried out at least three times, and the results were expressed by the mean ± standard deviation (SD). Students’ Additional file 1: Table S1. Detailed information of RimR2 and some polyene macrolide biosynthesis regulators from other Streptomyces species in phylogenetic tree.Additional file 2: Figure S1. Construction of mutant S. rimosus M527-ΔrimR2. Map of plasmid pWHU2653- ΔrimR2. The sgRNA consists of the 20 nt target gene specific guide sequence of S. rimosus M527 (green) and the invariant scaffold RNA (yellow). Light blue parallelograms connect the identical UHA and DHA sequences on pWHU2653 and the S. rimosus M527 chromosome where homologous recombination can take place.Additional file 3: Figure S2. PCR verification of the mutant S. rimosus M527-ΔrimR2. M: DL5000 DNA Marker. Lane 1, The PCR products of 2.8-kb rimR2 gene were amplified by using the primers PrimR2-F1/R1 from WT strain S. rimosus M527; Lane 2, The PCR products of 6.8-kb cassette containing 2.8-kb rimR2 gene and its 2.0-kb upstream and 2.0-kb downstream fragment were amplified by using the primers P1/P4 from S. rimosus M527; Lane 3-5, The PCR products of rimR2 gene were amplified by using the PrimR2-F1/R1 from three randomly mutant strains M527-ΔrimR2; Lane 6-8, The PCR products of 4.0-kb cassette containing 2.0-kb upstream and 2.0-kb downstream fragment were amplified by using the P1/P4 from three randomly mutant strains M527-ΔrimR2.Additional file 4: Figure S3. HPLC analysis of rimocidin production in the WT strain S. rimosus M527, in mutant S. rimosus M527-ΔrimR2, and in the complemented strain S. rimosus M527-ΔrimR2/pSET152::rimR2, and control strain S. rimosus M527/pSET152.Additional file 5: Figure S4. Purification and elution of RimR2 protein. M: Protein Marker; Lane 1, Purified His6-tagged RimR2 protein after affinity nickel-NTA column. Lanes 2-4, Eluted RimR2 protein with 250 mM, 300mM, 500mM imidazole.Additional file 6: Figure S5. Construction of recombinant plasmids for over-expression of rimR2 gene with different promoters.Additional file 7: Figure S6. Phenotypic verification of recombinant strains recombinant strains harboring over-expression of rimR2 gene. Recombinant strains could grow on 2CMC agar medium containing 300 μg/ml apramycin, while control strain S. rimosus M527 did not. 2CMC agar medium was incubated at 28 °C for 4 days.Additional file 8: Figure S7. PCR analysis of apramycin (apr) gene from recombinant strains harboring over-expression of rimR gene. DL DNA 2000 marker was used (M). Lane 1: PCR product of apr gene from S. rimosus M527(negative control); lane 2: PCR product of apr gene from plasmid pSET152(positive control); lane 3-5: PCR product of apr gene from recombinant strains S. rimosus M527-ER; lane 6-8: PCR product of apr gene from recombinant strains S. rimosus M527-KR; lane 9-11: PCR product of apr gene from recombinant strains S. rimosus M527-NR; lane 12-14: PCR product of apr gene from recombinant strains S. rimosus M527-21R; lane 15-17: PCR product of apr gene from recombinant strains S. rimosus M527-57R.Additional file 9: Figure S8. HPLC analysis of rimocidin isolated from fermentation extracts of the recombinant strains S. rimosus M527-KR, S. rimosus M527-NR, S. rimosus M527-ER, S. rimosus M527-21R, S. rimosus M527-57R and WT strain S. rimosus M527.Additional file 10: Figure S9. Construction of recombinant plasmids of overexpression of rimR1/rimR2/rimR3 /rimR4 gene with permE* promoter.Additional file 11: Figure S10. Detection and comparison of rimocidin production (a) and cell dry weight (b) of WT strain S. rimosus M527(●), recombinant strains M527-R1(■), M527-R2(▲), M527-R3(▼), and M527-R4(◆) in shake-flask culture experiment.Additional file 12: Figure S11. Detection and comparison of antifungal activities of WT strain M527, recombinant strains M527-R1, M527-R2, M527-R3, and M527-R4 against F. oxysporum f. sp. cucumerinum. Spore suspension (500 μl) of F. oxysporum f. sp. cucumerinum (1×106 cfu ml-1) was spread and inoculated on PDA medium at 28 °C for 1 d. A agar block (4 mm in diameter) containing actively growing WT strain M527, three random recombinant strains M527-R1(a), M527-R2(b), M527-R3(c), and M527-R4(d) was aseptically placed on aforementioned PDA medium containing pathogenic fungus at 28 °C for 3-4 d. The diameter of inhibition zone was measured as antagonistic activity. Plant-pathogenic fungus F. oxysporum f. sp. cucumerinum was used as indicator strain in antifungal activities assay.Additional file 13: Figure S12. Phylogenetic tree of RimR1 and other polyene macrolide biosynthesis regulators (PAS-LuxR).Additional file 14: Figure S13. Phylogenetic tree of RimR2, RimR3, RimR4 and other polyene macrolide biosynthesis regulators (LAL).Additional file 15: Table S2. The primers used for deletion or expression of rimR2 gene in this study.Additional file 16: Table S3. The primers used for EMSA assay in this study.


Results indicated that LPS decreased the villus height (p < 0.0001), increased the crypt depth (p < 0.05), and lowered the villus height to crypt depth ratio (p < 0.0001), while sodium acetate/sodium butyrate supplementation caused a significant increase in the villus height (p < 0.001), decrease in the crypt depth (p < 0.01), and increase in the villus height to crypt depth ratio (p < 0.001), especially. In mice treated with LPS, it was found that the serum level of IL-1β, TNF-α (p < 0.001), and MDA (p < 0.01) was significantly higher; however, sodium acetate/sodium butyrate supplementation significantly reduced IL-1β (p < 0.001), TNF-α (p < 0.01), and MDA (p < 0.01), respectively. A total of 19 genera were detected among mouse groups; LPS challenge decreased the abundance of Lactobacillus, unidentified F16, unidentified_S24-7, Adlercreutzia, Ruminococcus, unclassified Pseudomonadales, [Ruminococcus], Acetobacter, cc 1, Rhodococcus, unclassified Comamonadaceae, Faecalibacterium, and Cupriavidus, while increased Shigella, Rhodococcus, unclassified Comamonadaceae, and unclassified Pseudomonadales in group L. Interestingly, sodium acetate/sodium butyrate supplementation increased Lactobacillus, unidentified F16, Adlercreutzia, Ruminococcus, [Ruminococcus], unidentified F16, cc 115, Acetobacter, Faecalibacterium, and Cupriavidus, while decreased Shigella, unclassified Enterobacteriaceae, unclassified Pseudomonadales, Rhodococcus, and unclassified Comamonadaceae. LPS treatment upregulated the expressions of ZO-1 (p < 0.01) and NLRP3 (p < 0.0001) genes in mice; however, sodium acetate/sodium butyrate solution supplementation downregulated the expressions of ZO-1 (p < 0.05) and NLRP3 (p < 0.05) genes in treated mice. Also, the LPS challenge clearly downregulated the expression of Occludin (p < 0.001), Claudin (p < 0.0001), and Caspase-1 (p < 0.0001) genes, while sodium acetate/sodium butyrate solution supplementation upregulated those gene expressions in treated groups. The present study revealed that sodium acetate/sodium butyrate supplementation alleviated LPS-induced diarrhea in mice via enriching beneficial bacterium and decreasing pathogens, which could regulate oxidative damages and inflammatory responses via NLRP3/Caspase-1 signaling. The current results may give insights into the prevention and treatment of diarrhea.Diarrhea is a word-widely severe disease coupled with gastrointestinal dysfunction, especially in cattle causing huge economic losses. However, the effects of currently implemented measures are still not enough to prevent diarrhea. Previously we found that dropped short-chain fatty acids in diarrhea yaks, and butyrate is commonly known to be related to the epithelial barrier function and intestinal inflammation. However, it is still unknown whether sodium acetate/sodium butyrate could alleviate diarrhea in animals. The present study is carried out to explore the potential effects of sodium acetate/sodium butyrate on lipopolysaccharide-induced diarrhea in mice. Fifty ICR mice were randomly divided into control (C), LPS-induced (L), and sodium acetate/sodium butyrate -treated groups. Serum and intestine samples were collected to examine inflammatory cytokines, antioxidant levels, relative gene expressions  Diarrhea is a severe disease coupled with gastrointestinal dysfunction that has a global impact on fertility rate, milk production, and immunity in livestock . NowadayGut microflora is composed of millions of microorganisms that contribute remarkably to physiological processes, i.e., functions of nutrition absorption, metabolism, and immunity of the host by producing various metabolites . The anaLactobacillus plantarum alleviated diarrhea in a previous study by balancing gut microbiota and regulating SCFAs , and after 24 hours mice were euthanized to collect serum, small intestine, and rectum samples  were purchased from Qing Long Shan Dong Wu Fan Zhi . After 30 days of rearing, mice were randomly divided into five groups, namely control (C), LPS (L), and treatment groups . Group D (400: 200), B (300:300), and A (200: 400) were treated with 600 mg/kg sodium acetate/sodium butyrate solution  samples . All aniDuodenum, jejunum, and ileum samples from all the groups were preserved in 4% paraformaldehyde for at least 48 hrs and then processed for commercial H&E staining . On an Olympus CX23 microscope with an integrated digital imaging analysis system, histological slices were examined . The villus height and crypt depth were measured as depicted in the previous study .via commercial assay kits . The concentration of cytokines including interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), and tumor necrosis factor-alpha (TNF-α) was measured by using commercial ELISA kits .Blood samples of mice were centrifuged at 4,000 g for 10 min and stored at –20°C for future analysis. Antioxidant capacity was examined by detecting the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-px), total anti-oxidation capacity (T-AOC), and malondialdehyde (MDA) by utilizing commercial assay kits . Meanwhile, the NO concentrations were determined n = 4), L (n = 4), and A (n = 4) were extracted utilizing the fast DNA Stool Mini Kit  according to the manufacturer’s specifications. The quantity and quality of all extracted DNA samples were examined by using NanoDrop 2000 UV-vis spectrophotometer  and agarose gel electrophoresis, respectively. Gene amplification of bacterial 16S rRNA gene was performed using the V3–V4 regions primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Then all amplicon products were purified by employing Vazyme VAHTSTM DNA Clean Beads  and quantified using the QuantiFluor™-ST . At last, all samples were sequenced by using the Illumina MiSeq platform  with MiSeq Reagent Kit v3.The total microbial genomic DNA was extracted from the rectum contents of each mouse. The samples from groups C (1 (via mafft (2 (via ggtree3. Krona species composition map was generated via KronaTools (v2.7)4 (via PERMANOVA and Adonis in QIIME2 (2019.4). Venn5, heatmap, metagenomeSeq, LEFSe 4 . Signifiq, LEFSe , OPLS-DAq, LEFSe , and ranq, LEFSe  was perfq, LEFSe  using Mevia denaturing formaldehyde gel electrophoresis and NanoDrop 2000 analyzer  to validate their integrity and concentrations, respectively. Then commercial SuperScript™IV first strand cDNA synthesis kits  were used for translating RNA samples into cDNA under the guidance of the manufacturer’s specifications. Finally, qRT-PCR for all groups was carried out by using 25 uL of reaction mixtures consisting of 2 uL of intestinal tissues cDNA, 12.5 uL of Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix , 2uL of primers, and 8.5 uL nuclease-free water, then the procedure was performed in the StepOnePlus™ Real-Time PCR System . All sample reactions were repeated three times and the method of 2–ΔΔCT was utilized for calculating gene relative quantification. All primer pairs used in the present study were synthesized by Sangon Biotech (China) and are shown in Intestinal tissue RNA extraction from all mouse groups was performed by utilizing TRIzol reagent . All of the RNA samples were examined via ANOVA, Student’s t-test, Kruskal–Wallis, and Dunn’s test via IBM SPSS (22.0) software. Data presented as means ± SD and statistically significant are considered when P < 0.05.All the generated data were evaluated via H&E staining and found that LPS caused a decrease in the villus height (p < 0.0001), an increase in the crypt depth (p < 0.05), and it also lowered the villus height to crypt depth ratio (p < 0.0001). Whereas, sodium acetate/sodium butyrate supplementation resulted in a significant increase in the villus height (p < 0.001), decrease in the crypt depth (p < 0.01), and increase in the villus height to crypt depth ratio (p < 0.001), especially in the mice of groups B and A (The mice were weighed on a daily basis and the weight of the mice in group A was slightly higher than mice in other groups . The dia B and A .p < 0.05), while there was no marked difference between group L and treated groups D, B, and A, respectively. In group C, the serum level of mice induced by LPS was found prominently high for IL-1β (p < 0.001), TNF-α (p < 0.001), and MDA (p < 0.01), respectively. However, in the serum of groups D, B, and A, sodium acetate/sodium butyrate supplementation caused a remarkable decrease in IL-1β (p < 0.001), TNF-α (p < 0.01), and MDA (p < 0.01), respectively  and non-singleton (p < 0.05) was found between groups C and L (p < 0.05) , ANOSIM (p < 0.05), and PERMDISP (p < 0.05), respectively  . Beta diectively . UPGMA aectively . Signifiectively .Proteobacteria (83.02%) and Bacteroidetes (13.00%). While Firmicutes (83.36%), Bacteroidetes (12.80%), Firmicutes (45.35%), and Proteobacteria (46.88%) were the main phyla in groups C and A. At the Class level, the main classes were Gammaproteobacteria (81.28%) and Bacteroidia (12.99%) in group L, while Bacilli (74.72%), Bacteroidia (12.80%), Clostridia (8.08%), Gammaproteobacteria (45.90%), Bacilli (44.04%), and Bacteroidia (6.80%) were the dominant classes in groups C and A, respectively. At the order level, the primary classes were Enterobacteriales (55.60%), Pseudomonadales (25.65%), and Bacteroidales (12.99%), while Lactobacillales (72.68%), Bacteroidales (12.80%), Clostridiales (8.08%), Lactobacillales (43.52%), Enterobacteriales (41.07%), and Bacteroidales (6.80%) were the staple orders in groups C and A, respectively. At the family level, the dominating families in group L were Enterobacteriaceae (55.59%), Pseudomonadaceae (25.47%), and Bacteroidaceae (9.09%), while the main families were Lactobacillales (72.47%) and S24-7 (12.50%) in group C, whereas Lactobacillales (43.35), Enterobacteriaceae (41.07%), and Bacteroidaceae (4.84%) in group A, respectively. At the genus level, the major genera in group L were Shigella (54.62%), Pseudomonas (25.42%), and Bacteroides (9.09%), while Lactobacillus (72.45%), unidentified_S24-7 (12.50), Lactobacillus (43.34%), Shigella (40.71), and Bacteroides (4.84%) were the dominating genera in groups C and A , Bacteroides (9%), and Pseudomonas (8%) in group L, while Shigella (56%), Pseudomonas (26%), and Bacteroides (9%) in group A, and unidentified S34-7 (45%), unidentified Clostridia (15%), Lachnospiraceae (7%), and Turicibacter (7%) in group C, respectively  projected on the coordinate axis was clearly farther than groups A (light orange) and C (lavender), which revealed a difference between group L, and groups A and C, respectively  OTUs were shared in groups C and L, while 335 (11.96%) OTUs were shared in groups C and A . Then ASm genera . ASV/OTUs genera . It is d C and A . PCA anaectively . Also, Oectively .Bacteroidales and Lactobacillales were found significantly on the upside of the broken lines with decreased ASV 102 (p < 0.05), ASV 23 (p < 0.05), ASV 14 (p < 0.05), and ASV 53 (p < 0.05), increased ASV 73 (p < 0.05), ASV 1 (p < 0.01), ASV 194 (p < 0.05), ASV 50 (p < 0.05), ASV 18 (p < 0.01), and ASV 5 (p < 0.001) between groups C and A. Different genera like Bacteroidales, Lactobacillales, Clostridiales, Burkholderiales, Desulfovibrionales, Enterobacteriales, and Pseudomonadales were found significantly in groups C and L, with 107 decreased ASV and 86 increased ASV , family S24-7, order Clostridiales, class Clostridia, genus Gemella, order Gemellales, family Gemellaceae, phylum Tenericutes, class Mollicutes, family Lachnospiraceae, order RF39, family F16, class TMT-3, order CW040, phylum TM7, and genus Ruminococcus in group C (lavender), genus Faecalibacterium, family Acetobacteraceae, and order Rhodospirillales in group A (light orange)  .Clostridium, Ruminococcus, Lactococcus, Gemella, etc. Network analysis revealed that there were more edges between groups A and C than between groups L and C, which inferred a higher similarity between groups A and C , unidentified F16 (p < 0.0001), Adlercreutzia (p < 0.01), Ruminococcus (p < 0.05), [Ruminococcus] (p < 0.05), Acetobacter (p < 0.05), cc 115 (p < 0.05), and Cupriavidus (p < 0.05) in group L was obviously lower than group C, respectively. While Shigella (p < 0.05) was prominently higher in group L than in group C. Unidentified_S24-7 and unclassified RF39 were significantly higher in group C than in groups A (p < 0.05) and L (p < 0.05), respectively, while unclassified Enterobacteriaceae was significantly lower in group C than in groups A (p < 0.05) and L (p < 0.05), respectively. The abundance of unclassified Pseudomonadales (p < 0.05), Rhodococcus (p < 0.05), unclassified Comamonadaceae (p < 0.05), and Lysobacter (p < 0.05) in group L was conspicuously higher than group A, respectively. The abundance of Faecalibacterium in group L was clearly lower than groups A (p < 0.05) and L (p < 0.01), respectively. The abundance of Gluconacetobacter in group A was prominently higher than groups L (p < 0.05) and C (p < 0.01), respectively. The abundance of unclassified Bradyrhizobiaceae in group C was clearly higher than in group A (p < 0.05)  .In summary, different analysis methods include relative abundance taxa, classification levels tree diagram, GraPhlAn evolutionary tree diagram, Krona species composition diagram, Venn Diagram, Genera composition heatmap, PCA, OPLS-DA, metagenomeSeq analysis, LEfSe analysis, and random forests and network analyses demonstrated that LPS challenge changed microbiota composing in mice; however, sodium acetate/sodium butyrate supplementation could partly restore the gut microbiota in animals.p < 0.05), 9 (p < 0.01), and 20 (p < 0.001) significant different pathways between groups C and L, 37 (p < 0.05), 27 (p < 0.01), and 53 (p < 0.001) significant different pathways between groups A and C, respectively (p < 0.05), 15 (p < 0.01), and 51 (p < 0.001) significant different pathways between groups C and L, while one (p < 0.001) significant different pathway between groups A and C was examined  and NLRP3 (p < 0.0001) in mice of group L; however, sodium acetate/sodium butyrate solution supplementation downregulated the expression of ZO-1 (p < 0.05) and NLRP3 (p < 0.05) genes in treated mice. Furthermore, change in LPS clearly downregulated the expression of Occludin (p < 0.001), Claudin (p < 0.0001), and Caspase-1 (p < 0.0001) genes in group L, while sodium acetate/sodium butyrate solution supplementation upregulated those gene expressions in treated groups of D, B, and A (Relative gene expression of Zonula occludens 1 (ZO-1), Occludin, Claudin, Caspase-1, and NLRP3 were detected by employing qRT-PCR. LPS induction prominently upregulated the expression of ZO-1 , Phenylobacterium, and Aminobacter were positively related to inflammatory cytokines. Pseudomonas, Faecalibacterium, Gluconacetobacter, Rhodococcus, (Clostridium), and Gluconobacter were positively related to tight junction proteins, while Adlercreutzia, Candidatus Arthromitus, Lactococcus, Ruminococcus, Coprococcus, Acinetobacter, cc_115, Gemella, Anaeroplasma, Phenylobacterium, Lysobacter, and W22 were negatively related to tight junction proteins. Adlercreutzia, Ruminococcus, Coprococcus, cc_115, Roseburia, Gemella, and Anaeroplasma were negatively related to expressions of Caspase-1 and NLRP3 , intestine morphology indices, inflammatory cytokines, oxidative indices, and gene expressions was performed through Statistical Analysis System. Results showed that nd NLRP3 .Though various kinds of measures have been implemented so far to fight against diarrhea, there is still a long way to go . A recenIn the present study, mice were supplemented with sodium acetate/sodium butyrate before inducing diarrhea by utilizing LPS. Serve damage to intestines particularly jejunum, ileum, cecum, and colon was examined , which wTo explore the potential mechanisms, we detected the gene expressions of tight junction proteins in intestines. Among them Occludin and Claudins were recognized as important components of intestinal permeability . The expp < 0.01) was detected in group L; however, there was a significant decrease in MDA (p < 0.01) levels in sodium acetate/sodium butyrate supplemented groups D, B, and A, respectively (p < 0.0001) in mice in group L; however, sodium acetate/sodium butyrate solution supplementation downregulated the expression of NLRP3 (p < 0.05) genes in treated mice. Also, the LPS challenge clearly upregulated the Caspase-1 (p < 0.0001) gene in group L, while sodium acetate/sodium butyrate solution supplementation downregulated it in treated groups D, B, and A (p < 0.001) were found in group C, which was consistent with previous results found higher inflammatory factors and upregulation of NLRP3 in HUVEC cells , Acetobacter, cc 1, Rhodococcus, unclassified Comamonadaceae, Faecalibacterium, and Cupriavidus, while increased Shigella, Rhodococcus, unclassified Comamonadaceae, and unclassified Pseudomonadales in group L. Interestingly sodium acetate/sodium butyrate supplementation increased Lactobacillus, unidentified F16, Adlercreutzia, Ruminococcus, (Ruminococcus), unidentified F16, cc 115, Acetobacter, Faecalibacterium, and Cupriavidus, while decreased Shigella, unclassified Enterobacteriaceae, unclassified Pseudomonadales, Rhodococcus, and unclassified Comamonadaceae. Lactobacillus genus bacteria are probiotic microorganisms that have beneficial effects on to host .KL and QK: research idea, methodology, visualization, and supervision. XC, QK, XZ, CZ, PH, and HL: reagents, materials, and analysis tools. KL: writing—original draft and preparation. MK, ZB, II, HA, QS, and KL: writing—review and editing. All authors contributed to the article and approved the submitted version.


Limited data exist regarding longer term antibody responses following three-dose coronavirus disease 2019 (COVID-19) vaccination, and the impact of a first SARS-CoV-2 infection during this time, in people with HIV (PWH) receiving suppressive antiretroviral therapy (ART). We quantified wild-type-specific, Omicron BA.1-specific and Omicron BA.5-specific responses up to 6 months post-third dose in 64 PWH and 117 controls who remained COVID-19-naive or experienced their first SARS-CoV-2 infection during this time.Longitudinal observational cohort.We quantified wild-type-specific and Omicron-specific anti-Spike receptor-binding domain IgG concentrations, ACE2 displacement activities and live virus neutralization at 1, 3 and 6 months post-third vaccine dose.Third doses boosted all antibody measures above two-dose levels, but BA.1-specific responses remained significantly lower than wild-type-specific ones, with BA.5-specific responses lower still. Serum IgG concentrations declined at similar rates in COVID-19-naive PWH and controls post-third dose . Antibody function also declined significantly yet comparably between groups: 6 months post-third dose, BA.1-specific neutralization was undetectable in more than 80% of COVID-19 naive PWH and more than 90% of controls. Breakthrough SARS-CoV-2 infection boosted antibody concentrations and function significantly above vaccine-induced levels in both PWH and controls, though BA.5-specific neutralization remained significantly poorer than BA.1 even post-breakthrough.Following three-dose COVID-19 vaccination, antibody response durability in PWH receiving ART is comparable with controls. PWH also mounted strong responses to breakthrough infection. Due to temporal response declines, however, COVID-19-naive individuals, regardless of HIV status, would benefit from a fourth dose within 6 months of their third. In British Columbia (BC), Canada, third doses of coronavirus disease 2019 (COVID-19) monovalent mRNA vaccines were introduced in November 2021, initially to individuals at risk of severe COVID-19 outcomes, including some people with HIV (PWH). Whether offered as part of a primary vaccine series or a ‘booster’, third doses help to maintain systemic immunity and enhance protection against infection by viral variants . DespiteLongitudinal monitoring of immune responses post-third dose in PWH is critical to inform the timing of future immunizations in this group. Though some data are available on initial immunogenicity to third COVID-19 vaccine doses in PWH –16, no sOur cohort was described previously . The preThis study was approved by the University of British Columbia/Providence Healthcare and Simon Fraser University Research Ethics Boards. All participants provided written informed consent.50/200 μl in the presence of serial two-fold plasma dilutions (1/20 to 1/2560) and added to target cells in triplicate. Viral cytopathic effects (CPE) were recorded 3 days post-infection. Neutralization was reported as the highest reciprocal dilution able to prevent CPE in all three wells. Partial or no neutralization at 1/20 dilution was considered below the limit of quantification (BLOQ) and coded as a reciprocal dilution of 10.Assays were performed as previously described ,19. IgG-U test (unpaired data) or Wilcoxon test (paired data). Relationships between continuous variables were assessed using Spearman's correlation. In participants who remained COVID-19-naive, multiple linear regression was used to investigate the relationship between HIV infection and vaccine-induced immune measures using a confounder model that adjusted for variables that could influence vaccine responses or that differed in prevalence between groups. For Omicron-specific neutralization at 6 months post-third dose, multiple logistic regression was used because of the high proportion of results BLOQ. Included variables were: HIV infection (controls as reference group), age (per year), sex at birth , ethnicity (nonwhite as reference), number of chronic conditions , dual ChAdOx1 as the initial regimen [mRNA or mixed (ChAdOx1/mRNA) regimen as the combined reference group] [P less than 0.05 considered statistically significant. Analyses were conducted using Prism v9.2.0 .Continuous variables were compared using the Mann–Whitney e group] ,17, thir+ T-cell counts of 645 [interquartile range (IQR) 473–958] cells/μl, and median nadir CD4+ T-cell counts of 225 (IQR 95–485) cells/μl, at enrolment. PWH were a median of 57 (IQR 42–65) years old and 90% men; controls were a median 47 (IQR 35–72) years old and 73% women. PWH had a higher proportion of white ethnicity  and more chronic health conditions [median 1 (IQR 1–3) compared with 0 (IQR 0–1) in controls]. More PWH (10%) than controls (<1%) received two doses of the recombinant viral vector ChAdOx1 vaccine as their initial immunization series. All third doses were monovalent mRNA vaccines, either BNT162b2 (30 μg) or mRNA-1273 (50 or 100 μg). Most PWH (69%) and controls (62%) received mRNA-1273, where, per local guidelines, all adults aged at least 70 years were eligible for a 100 μg mRNA-1273 dose, as were PWH who met one or more of the following criteria: age at least 65 years, prior AIDS-defining illness, prior CD4+ T-cell count less than 200 cells/μl, prior CD4+ T-cell fraction 15% or less, any plasma HIV load greater than 50 copies/ml in 2021, or perinatally acquired HIV [Characteristics of the 64 PWH and 117 controls, all of whom remained COVID-19-naive until at least 1 month post-third dose, are shown in Table P = 0.4). Though SARS-CoV-2 variant information is unavailable for individual infections, most were likely Omicron BA.1 or BA.2 based on local epidemiology at the time [A total of 24 (38%) PWH and 45 (39%) controls experienced their first SARS-CoV-2 infection between 1 and 6 months post-third dose (one control experienced two infections during this period ). Based the time .10 BAU/ml in COVID-19-naive PWH and 3.67 (IQR 3.50–3.86) log10 BAU/ml in controls, respectively (P = 0.5). Following this, wild-type-specific IgG responses declined comparably in COVID-19-naive PWH and controls. At 3 months, wild-type-specific IgG responses in PWH and controls declined to a median 3.48 (IQR 3.12–3.75) and 3.40 (IQR 3.21–3.61) log10 BAU/ml, respectively (P = 0.5), while by 6 months, responses had declined to a median 2.96 (IQR 2.62–3.38) and 3.06 (IQR 2.82–3.24) log10 BAU/ml in PWH and controls, respectively (P = 0.4) , whereas those in controls had declined to significantly lower than post-second dose levels (P < 0.0001)  . One month post-third dose, for example, BA.1-specific IgG concentrations were 2.96 (IQR 2.71–3.32) log10 BAU/ml in PWH, which was 0.74 log10 BAU/ml lower than wild-type-specific concentrations at this time. By 6 months, BA.1-specific IgG concentrations had declined to 2.47 (IQR 2.14–2.59) log10 BAU/ml in PWH, which was 0.49 log10 BAU/ml lower than wild-type-specific concentrations at this time.Similarly, Omicron BA.1-specific IgG responses were comparable in COVID-19-naive PWH and controls at all post-third dose time points , as was male sex (P = 0.012 for BA.1-specific responses) (Supplementary Table 1). In fact, adjusted IgG concentrations were slightly higher in PWH compared with controls, though this was not statistically significant. Of note, receipt of an mRNA-1273 third dose (rather than BNT162b2) was associated with stronger wild-type-specific IgG responses (P = 0.029), though not BA.1-specific responses (P = 0.13), 6 months post-third dose. Among PWH, we also observed no significant relationship between most recent or nadir CD4+ T-cell count and either wild-type-specific or BA.1-specific IgG concentrations at this time .We next performed multivariable analyses adjusting for sociodemographic, health and vaccine-related variables to identify variables associated with wild-type-specific and BA.1-specific IgG concentrations at 6 months post-third dose in the COVID-19-naive subgroup. These analyses revealed that a higher number of chronic health conditions – but not HIV infection – was associated with poorer IgG responses at this time . Estimated BA.1-specific IgG half-lives were also comparable, at a median 71 (IQR 49–104) days for PWH compared with 74 (IQR 59–90) days in controls (P = 0.8). Multivariable analyses confirmed that HIV infection was not associated with wild-type-specific or BA.1-specific IgG half-lives post-third dose (Supplementary Table 2). Among COVID-19-naive PWH, we initially observed a weak inverse correlation between recent CD4+ T-cell count and IgG half-life, but this was not significant after excluding an outlier with a long (>200 day) half-life .We estimated the half-lives of wild-type-specific IgG following three-dose vaccination to be a median 66 (IQR 47–89) days in COVID-19-naive PWH compared to 72 (IQR 54–96) days in controls, a difference that was not statistically significant , which was 0.27 log10 BAU/ml higher than at 1 month post-third dose (P < 0.0001). Importantly, the magnitude of these ‘hybrid’ IgG responses was comparable between PWH and controls at all post-infection time points tested . Notably, while most participants experienced a marked boost in antibody levels following SARS-CoV-2 infection, IgG responses in a minority of PWH and controls remained constant or even declined post-infection , while by 6 months, activities had decreased to a median 87% (IQR 67.5–98.1) in PWH versus 93.6% (IQR 81.5–97.8) in controls (P = 0.3), levels that were comparable or lower than after two-dose vaccination   compared with 99.1% (IQR 97–99.6%) in controls , but these responses also declined similarly in COVID-19-naive PWH and controls , to 30.8% (IQR 23.0–54.1) in PWH and 25.9% (IQR 12.1–52.2) in controls (P = 0.3), where the latter values were significantly lower than those observed after two-dose vaccination .BA.1-specific responses remained significantly lower than wild-type-specific responses at all time points . There was also no evidence that a low recent or nadir CD4+ T-cell count was associated with lower wild-type-specific or BA.1-specific ACE2 displacement activities 6 months post-third dose in COVID-19-naïve PWH . This was consistent with prior observations at 1 month post-third dose, which we attributed to the observation that PWH with low nadir CD4+ T-cell counts were eligible for the higher (100 μg) third mRNA-1273 dose [Multivariable analyses confirmed that, among COVID-19-naive participants, HIV infection was not associated with either wild-type-specific or BA-1-specific ACE2 displacement activities at 6 months post-third dose (Supplementary Table 3). Rather, having received a mRNA-1273 third dose was the only independent correlate of stronger wild-type-specific ACE2 displacement activity at this time (273 dose .P < 0.0001), where activities at 6 months in this group were overall significantly greater than peak responses induced by three-dose vaccination (both P < 0.0001) . It is again notable, however, that a minority of PWH and controls did not show appreciable increases in this activity following infection  , while by 6 months, neutralization had declined to a median 80 (IQR 35–160) in PWH and 40 (IQR 20–80) in controls (P = 0.006), values that were below the levels originally elicited by two-dose vaccination (both groups P ≤ 0.01). As reported previously [One month post-third dose, wild-type-specific neutralization activity in PWH  was slightly higher than in controls , though responses in PWH were not further impaired compared with controls. In fact, 1 month post-third dose, BA.1-specific neutralization was higher in PWH compared with controls: a median 60 (IQR 25–160) and 40 (IQR 20–40), respectively (P = 0.0005), though these values were five-fold to eight-fold lower than corresponding wild-type-specific responses. BA.1-specific neutralization subsequently declined rapidly; at 3 months, this was BLOQ in 67.4% of COVID-19-naive PWH and 80.5% of controls (P = 0.2), whereas at 6 months, this was BLOQ in 82.4% of PWH and 92.2% of controls (P = 0.2), levels that were significantly lower than peak responses after two-dose vaccination . In multivariable analyses, older age – but not HIV infection – was the only significant correlate of poorer BA.1-specific neutralization among COVID-19-naive individuals at 6-month post-third dose . We also observed no significant associations between CD4+ T-cell parameters and either wild-type-specific or BA-1-specific neutralization at 6 months among COVID-19-naive PWH .BA.1-specific neutralization was significantly lower than wild-type responses at all timepoints for all groups  .By contrast, individuals who experienced breakthrough infection showed significantly stronger wild-type-specific and BA.1-specific neutralization compared with their COVID-19-naive counterparts at 3 and 6 months , and 16-fold lower than that for wild-type   and wild-type   .Since the emergence of Omicron BA.1, even more immune evasive variants have developed, including Omicron BA.5 that has dominated globally –26. We, Our results demonstrate that antibody response durability following three-dose COVID-19 vaccination in COVID-19-naive PWH receiving suppressive ART is comparable to controls without HIV. As we reported previously ,17, initBy contrast, almost all participants who experienced their first SARS-CoV-2 infection (presumably BA.1 or BA.2 ) post-th+ T-cell counts and/or who are not receiving suppressive ART. Indeed, results from several studies indicate that PWH with CD4+ T-cell counts below 500 cells/μl mount weaker responses to the first [Our study has several limitations. Our observations may not be generalizable to PWH with low CD4he first –32 and she first –38 doseshe first , future he first –44.In conclusion, our observations confirm the humoral immune benefits of third COVID-19 vaccine doses in PWH receiving suppressive ART, and further reveal that the durability of third-dose responses is comparable to that in persons without HIV. Nevertheless, regardless of HIV status, individuals who remain COVID-19-naive will benefit from a fourth dose within 3–6 months of their third dose, as antibody concentrations and neutralization activities declined markedly over time in this group, and the ability of vaccine-induced responses to neutralize the dominant Omicron BA.5 variant were even poorer than BA.1. By contrast, the majority of individuals who experienced their first SARS-CoV-2 infection post-third vaccine dose showed significantly higher antibody activities than those induced by vaccination alone . These observations suggest that a slightly delayed fourth dose (e.g. to 3–6 months following infection) would optimally benefit this group. Further studies of hybrid response durability are required, as are direct comparisons with immune responses elicited by a fourth dose in COVID-19-naive individuals, particularly in light of new bivalent formulations that include wild-type and Omicron Spike antigens.This work is dedicated to the memory of our friend and colleague Hesham Ali who sadly passed away in July 2022. We thank the phlebotomists and laboratory staff at the BC Centre for Excellence in HIV/AIDS, the Hope to Health Research and Innovation Centre, St. Paul's Hospital, and Simon Fraser University for assistance. Above all, we thank the participants, without whom this study would not have been possible.Author contributions: M.A.B. and Z.L.B. are co-principal investigators and conceived the study, with C.T.C., C.C., A.H.A., V.L., M.H., M.G.R., R.G., S.G., J.S.G.M., M.H. and M.H. additionally contributing to study design and development. M.A.B., Z.L.B., M.G.R., A.H.A., C.T.C. and C.C. obtained project funding. M.A.B., Z.L.B., H.R.L., F.M. and P.K.C. designed experiments. H.R.L., R.M., P.K.C., Y.S., F.Y., S.S., E.B., N.M.-G., S.D., M.C.D., R.K., S.E., L.Y., B.G., F.H.O., G.U., J.T., P.S., L.B. and C.J.B. contributed to specimen collection, data collection, data curation and/or data analysis. H.R.L., Y.S., D.H., M.L.D., J.S., M.N., M.G.R., M.A.B. and Z.L.B. supervised the research, laboratory assays and/or contributed to project or cohort management. Z.L.B. performed statistical analyses with support from C.J.B.. N.P., C.F.L., M.G.R., W.D., C.J.B., and M.N. provided, generated and/or validated local Omicron isolates. H.R.L. wrote the first draft of the manuscript.Funding: this work was supported by funding from Genome BC, Michael Smith Health Research BC, and the BCCDC Foundation for Public Health through a rapid SARS-CoV-2 vaccine research initiative in BC award . It was also supported by the Public Health Agency of Canada (PHAC) through two COVID-19 Immunology Task Force (CITF) COVID-19 Awards . Additional funding was received from the Canadian Institutes for Health Research , the Coronavirus Variants Rapid Response Network , the Canada Foundation for Innovation through two Exceptional Opportunities Fund COVID-19 awards , a British Columbia Ministry of Health–Providence Healthcare Research Institute COVID-19 Research Priorities Grant (to C.J.B. and C.F.L.) and the CIHR Canadian HIV Trials Network (CTN) (to A.H.A.). F.M. is supported by a fellowship from the CIHR Canadian HIV Trials Network. M.L.D. and Z.L.B. hold Scholar Awards from Michael Smith Health Research BC. F.Y. and E.B. were supported by SFU Undergraduate Research Awards. M.D. is supported by a CIHR Canada Graduate Scholarships-Master's award. G.U. and F.H.O. are supported by PhD fellowships from the sub-Saharan African Network for TB/HIV Research Excellence (SANTHE), a DELTAS Africa Initiative (grant # DEL-15-006). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)'s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa's Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust (grant # 107752/Z/15/Z) and the UK government. The views expressed in this publication are those of the authors and not necessarily those of PHAC, CITF, AAS, NEPAD Agency, Wellcome Trust, the Canadian or UK governments or other funders.https://doi.org/10.1101/2022.11.03.22281912Posted history: this manuscript is posted to MedRxiv: There are no conflicts of interest.