Bone is capable of adjusting size, shape, and quality to maintain its strength, toughness, and stiffness and to meet different needs of the body through continuous remodeling. The balance of bone homeostasis is orchestrated by interactions among different types of cells (mainly osteoblasts and osteoclasts), extracellular matrix, the surrounding biological milieus, and waste products from cell metabolisms. Inorganic ions liberated into the localized microenvironment during bone matrix degradation not only form apatite crystals as components or enter blood circulation to meet other bodily needs but also alter cellular activities as molecular modulators. The osteoinductive potential of inorganic motifs of bone has been gradually understood since the last century. Still, few have considered the naturally generated ionic microenvironment’s biological roles in bone remodeling. It is believed that a better understanding of the naturally balanced ionic microenvironment during bone remodeling can facilitate future biomaterial design for bone tissue engineering in terms of the modulatory roles of the ionic environment in the regenerative process. A localized microenvironment in bone remodeling milieus is generated and maintained when ions and biological molecules are released during the demineralization and degradation of bone matrix by protons and proteases secreted by osteoclasts, respectively, and bone formation by osteoblasts . HoweverInspired by the abundance of elements in the biological system and the effects of nutritional deficiency or overload, therapeutic applications of bioinorganic ions have been explored for many years. For example, the platinum drug cisplatin has been used for cancer treatment, the gold drugs myocrisin and auranofin for rheumatoid arthritis treatment, silver compounds in the pharmaceutical industry for their antimicrobial properties, and lanthanides and some transition metals as radiopharmaceuticals and diagnostic agents ,6,7. MeaAmong all, calcium phosphates (CaPs) based on inorganic biomaterials are one of the most extensively studied types for bone grafting. They are composed of calcium ions and phosphate groups, which are omnipresent in the bloodstream or fixed in the bone mineral phase ,9,10. ThBone homeostasis is maintained in a series of highly complicated events orchestrated by: (a) interactions among different types of cells, mainly mesenchymal stem cells (MSCs), osteoprogenitor cells, osteoblasts, osteoclasts, and osteocytes, and (b) interactions of cells with extracellular matrix in a localized microenvironment, and (c) interactions of cells with components in surrounding biological milieus, such as organics , inorganics , as well as waste products from cell metabolism  9]. Nat. Nat9]. c-axis parallel to the direction of fibrils, with lengths of 30–50 nm, widths of 15–30 nm, and thicknesses of 2–10 nm [−), parallel to the c-axis, are positioned on the screw axes at every one-half of the unit cell, pointing in opposite directions to neighboring OH−s. Tetrahedral phosphate ions (PO43−), immobilized by calcium ions (Ca2+) interspersed among them, as well as marginal calcium ions (Ca2+), shared with neighbor unit cell. Notably, steric interference between adjacent OH−s in hexagonal hydroxyapatite unit cells could be overcome by vacancy or replacement of an OH− by impurity ions, the most likely event in organisms, or by conversion of hexagonal to monoclinic space group at high temperature, rearranging adjacent OH−s to a uniform direction [Bone mineral, known as biological apatite, is incorporated in collagen fibrils, arranged with a  2–10 nm . Biologiirection ,26.− (minor site) or PO43− (primary site) sites could be replaced by CO32−, forming type A and B carbonated hydroxyapatite, respectively. PO43− site could also be replaced by hydrolyzed phosphate (HPO42−) structure in mature bone, while OH− could be substituted with florin (F−), chlorin (Cl−) ion, or orthosilicic acid (SiO44−) structure [2+ is replaced by other metals, such as sodium (Na+), potassium (K+), magnesium (Mg2+), zinc (Zn2+), manganese (Mn2+), cobalt (Co2+), strontium (Sr2+), iron (Fe2+), copper (Cu2+). Ionic exchange in biological apatite alters crystalline structures, resulting in modified crystal size, growth rate, and properties. Compared with stoichiometric or geological apatite crystals, biological apatite crystals have smaller crystallite sizes, less ordered crystal structure, lower crystallinity, and higher solubility  material design with proper geometry, roughness, and porosity that facilitates bone growth by entrapping and concentrating growth factors or osteoprogenitor cells, (b) incorporation with growth factors, bioactive proteins or trace elements that would induce bone growth ,59. If oOsteoinductivity of demineralized bone matrix in different animals was reported in 1965, and osteogenic factors originally present in the matrix, specifically bone morphogenetic proteins (BMPs), were demonstrated later ,61. InorWith intrinsic properties of inorganic biomaterials to release ions, osteoinductive inorganic biomaterials can also be achieved via increased ion concentration in the localized microenvironment. In general, the liberation of ions from biomaterials is believed to enrich the ionic microenvironment, alter ion concentrations and local pH and get involved in bone formation by increasing the supersaturation of ions toward the deposition of hydroxyapatite or as molecular modulators to affect cell signaling and activities ,4,16,54.Inorganic ions are not only nutrients in the body but also have the potential as components in diagnostic or therapeutic agents to study or treat various diseases and metabolic disorders, explaining why they have great potential to affect bone regeneration to a similar extent as recombinant growth factors but free from safety issues ,23. For 2+ has been shown to correlate with multiple cellular activities of MSCs , osteoblasts , and osteoclasts  via a variety of intracellular signaling pathways in vitro [2+ is 1.8 mM, the same concentration supplied in culture media to maintain cell growth [2+ at a concentration <1.8 mM significantly impeded cell growth and osteogenic differentiation [2+ concentration, on the other hand, showed no additional promotive effect on cell growth but affected the extent of cell mineralization in a dose-dependent manner [2+ concentration is most likely to be sensed by the functional calcium-sensing receptor (CaSR) on the cell membrane, which is a member of the G-protein-coupled receptor (GPCR) superfamily [2+ concentration and modulating cell metabolism in many cells, such as parathyroid gland cells, kidney cells, bone cells, endothelial cells, and stem cells [2+ has been suggested to promote osteoblast proliferation and survival (2–4 mM), as well as differentiation (~5 mM), with elevated expression of several osteogenic markers such as type I collagen (Col-I), bone morphogenetic proteins (BMP), osteocalcin (OCN), etc., most likely via CaSR-mediated signaling pathways [2+ environment to affect cell survival [2+ level and controlling osteoblast cell fate, such as insulin-like growth factor (IGF)-II (required for the subsequent cell proliferation), or prostaglandin E2 (PGE2) produced by cyclooxygenase-2 (COX-2)  [2+ has also been shown to regulate cell morphology via cell-cell or cell-matrix interaction, enhancing the expression of angiopoietin-1 (Ang1) and angiogenesis [2+ has been shown to sequentially activate the PLC signaling pathway, followed by PLC-dependent translocation of nuclear factor-κB (NF-κB) from the cytoplasm to the nucleus of mature osteoclasts and consequently induce cell apoptosis to inhibit bone resorption [Extracellular Cain vitro ,164,165.l growth . The micntiation . Higher t manner . The fluerfamily ,164,165.em cells . In ostepathways ,170,171.pathways ,78. The pathways ,78. Othesurvival ,172,173.ocalcin) ,175. Morogenesis ,167,176.sorption ,79,80.2+ via ERK1/2- dependent signaling pathways and upregulates the expression of Fos-related antigens 1 and 2 (Fra-1/2) of activator protein-1 (AP-1) family in vitro [The homeostasis of phosphate in the body is maintained by the cooperation of the gut, bones, and kidneys and balanced by parathyroid hormones, but limited knowledge from the entry beyond regarding the sensing mechanism and consequent proposal for appropriate regulation cascade ,177. Thein vitro ,182,183.in vitro ,82. Howein vitro ,83. In oin vitro ,184. Thein vitro .ALP, OCN, and Col-I) expression and inducing early matrix mineralization in MSCs, and regulating osteogenic marker expression (runt-related transcription factor 2 (Runx2), and bone sialoprotein (BSP) at mRNA level, BMP-4, -6 and -7 at protein level) in osteoblasts [Boron (B), an essential micronutrient, is considered to play an important role in the maintenance of bone and osteogenesis. Several in vivo studies have shown that B ion deficiency could result in reduced osteogenesis, and B ion deprivation would inhibit bone formation, resulting in reduced bone volume and mechanical strength. Beneficial effects on bone micro-architecture and strength could be observed with the nutritional intake of B ,88,89,90eoblasts ,91,92.2+ deficiency causes abnormal bone formation with impaired quality as a co-factor of an enzyme, lysyl oxidase [2+ have attracted much attention in biomaterial fabrication [Copper (Cu) is an essential trace element required for the function of several important enzymes in the body, and it is necessary to maintain bone quality and strength . Cu2+ de oxidase . It prev oxidase ,94,95. Nrication ,97,98. Rrication .2+ channel ; Improved mineralization and elevated mechanical properties results from inhibited expression of nuclear factor of activated T cells, cytoplasmic 1 gene  [Gallium (Ga) is not an essential element in the body but positively affects bone formation with profound anti-resorptive activity . Ga ion tiation) ,101,102.tiation) .2+ is found to enhance the expression of the osteogenesis-related genes, production of extracellular matrix, and deposition of apatite crystal in undifferentiated MSCs and osteoblastic MSCs in vitro, possibly through the upregulation of hypoxia-inducible factor (HIF) and peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), respectively [2+ (up to 5 mM) competes with Ca2+ as an antagonist and forms an insoluble salt with pyrophosphate, causing mineralization defect and cell dysfunction [Magnesium (Mg), the second most abundant intracellular cation, stabilizes DNA and RNA structures and cell membranes and plays an essential role in maintaining the function of many enzymes as co-factors ,186. In ectively ,109,110.ectively ,109,110.function ,111,112.Iron (Fe) is an essential element for humans. In skeletal bone, Fe contributes to the homeostasis of bone, with evidence showing that: Fe deficiency causes an overall loss in bone mass and density, with impaired biomechanical strength ; Fe overALP, BMP), and accelerates collagen deposition [2+ exhibits an insulin-like effect, promoting angiogenesis and bone healing in vivo [Manganese (Mn), an essential cofactor for many enzymes, is required in many biological processes. In skeletal bone, Mn deficiency causes abnormal bone growth (such as stunted bone growth and osteoporosis), while Mn overload leads to impaired bone development and neurotoxicity ,117,118.position ,121,122. in vivo .Selenium (Se) is an essential trace element in humans. The level of Se is correlated with bone metabolism and maintenance (Kashin-Beck disease), as well as well-being and protection against aging-related diseases ,130,131.2, CaO, Na2O, and P2O5, was developed in the late 1960s [Silicon (Si), mainly found in the skeleton, is essential in bone metabolism ,188,189.te 1960s . Bioactite 1960s ,133,134.te 1960s ,139,140.te 1960s . In addite 1960s .2+ can activate the Wnt/β-catenin pathway or Ras/MAPK signaling pathway to upregulate the expression of osteogenic differentiation markers in cultured MSCs , facilitate calcium deposition and nodule formation, and promote in vivo bone formation [2+ (20 and 100 μg/mL) was found to disturb mineralization in rodent MSCs [Strontium (Sr), mostly stored in skeleton bone, can exert many effects on bone metabolism at cellular and tissue levels in vitro and in vivo ,193. Strormation ,143,144.ormation ,141,142.ormation . Additioent MSCs . In osteent MSCs ,149. Theent MSCs ,151.2+ released from the implant material has been shown to promote cell viability, osteoblastic marker gene expression , matrix maturation, calcium deposition, and nodule formation [2+ is also believed to participate in bone metabolism as a signaling molecule modulating osteoblast and osteoclast cellular activities in vitro and in vivo [2+ stimulates cell proliferation, differentiation, and mineralization by stimulating gene expression of various proteins associated with osteoblastic differentiation, such as type I collagen, ALP, OCN, OPN, and Runx2, and production of growth factors, such as IGF-1 or estrogen, related to enhanced cell proliferation [2+ acts as a potent inhibitor of resorptive activities [2+ in promoting bone formation and suppressing bone resorption are achieved via the inhibition of the activation of TNFα driven NF-κB pathway [Zinc  is an esormation . Zn2+ is in vivo ,157,158.feration ,160. In tivities . The mec pathway .2+ and Cr3+ into the peri-implant bone and cause progressive local osteolysis [2+ and Cr3+ are well-established in osteoblast-like cells with altered morphology, decreased cell number, proliferation, and cellular activities with decreased release of OCN and collagen type I, reduced ALP activity and calcium deposition, possibly due to altered redox state [2+ in promoting vascularization in bone tissue is still worth pursuing because vascularization is also a critical component in bone regeneration [In addition, the influence of some elements remains controversial due to conflicting results obtained in different studies, such as Fluorine (F), Lithium (Li), and Titanium (Ti). Some are being investigated due to observed positive effects in some therapeutical applications in bone diseases or biomaterial fabrication, such as Germanium (Ge), Niobium (Nb), and Vanadium (V) ,185. Somteolysis ,198,199.ox state ,201. Groox state . Neverthneration ,204,205.2+, PO43−, Mg2+, etc.) is degraded. There is a wealth of evidence revealing the osteoinductive potentials of many individual ions, but few consider the effect of the ionic microenvironment. The composition and interactions among components in the localized ionic environment during bone remodeling seemed challenging to investigate. Still, we believe the mystery will be unveiled with more emerging state-of-the-art techniques and a deeper understanding of related fields. Additionally, more fundamental research is needed to address the effective species and dosage during biomaterial fabrication in the future. Nevertheless, the beneficial effects of ions in bone tissue engineering will shed light on the design of future inorganic biomaterials for bone regeneration. An inorganic biomaterial that provides a balanced combination of inorganic ions in a controlled and sustained way will potentially generate a desired ionic environment to regulate bone cell functions, resulting in optimal tissue regeneration.A localized ionic environment is generated during bone remodeling, where the bone matrix containing organic molecules  and inorganic ions and ionic groups (Ca


Lung cancer is the second most common cancer in the world, being the first cause of cancer-related mortality. Surgery remains the only potentially curative treatment for Non-Small Cell Lung Cancer (NSCLC), but the recurrence risk remains high (30–55%) and Overall Survival (OS) is still lower than desirable (63% at 5 years), even with adjuvant treatment. Neoadjuvant treatment can be helpful and new therapies and pharmacologic associations are being studied. Immune Checkpoint Inhibitors (ICI) and PARP inhibitors (PARPi) are two pharmacological classes already in use to treat several cancers. Some pre-clinical studies have shown that its association can be synergic and this is being studied in different settings. Here, we review the PARPi and ICI strategies in cancer management and the information will be used to develop a clinical trial to evaluate the potential of PARPi association with ICI in early-stage neoadjuvant setting NSCLC. Lung cancer is the second most common cancer in the world, with an estimated incidence of 2.2 million new cases a year, being also the first cause of cancer-related mortality with approximately 1.8 million deaths a year ,4 AlthouThe treatment of NSCLC requires the consideration of different variables such as the stage of the disease, histological classification, Programmed Death Ligand-1 (PDL-1) expression, mutational status, and patient performance status. Currently, the NSCLC treatment is based on surgery, radiotherapy, chemotherapy, immunotherapy, and Tyrosine Kinase Inhibitors (TKI) for targeted molecular alterations, either in monotherapy or in combination. In the early stages (I-II), the elective treatment remains surgery either alone or followed by adjuvant treatment with chemotherapy alone or combined with radiotherapy. In locally advanced stage III disease T3-4, N2-N3), which occurs in approximately 30% of patients with NSCLC, most patients are non-surgical candidates and, currently, the standard of care treatment is chemoradiotherapy followed by maintenance immunotherapy -4, N2-N3,6. In paNotwithstanding the multiple recent advances in the treatment of advanced NSCLC, early stages remained with the same standard of care for decades . SurgeryIn recent years, several therapies, such as targeted therapy, immunotherapy, poly-ADP-ribose polymerase (PARP) inhibitors, and others, either alone or in combination, are being studied in the neoadjuvant setting some of which with favorable results already available ,16,17,18The theory of immune surveillance suggests that the immune system is able of destroying aberrant cells and preventing cancerous development. Some studies show that patients with compromised immune systems can have an increased risk of developing cancers but also that the cancerous cells can gain the ability to evade the immune response ,24,25,26It is known that Programmed Cell Death Protein 1 (PD-1) is highly expressed in Tumor-Infiltrating Lymphocytes (TILs) and that PD-1 Ligands  and Cytotoxic T Lymphocyte Associated protein 4 (CTLA-4) can be expressed in many cancerous cell types. PD-L1 can be expressed or upregulated in cancerous cells by two general mechanisms, namely innate resistance and adaptative immune resistance. In innate immune resistance, PD-L1 expression is upregulated by constitutive oncogenic signaling through aberrant activation of the PI3K-AKT pathway or chromosomal alterations and amplifications. PD-L1 expression in tumor cells can also be induced and/or upregulated as a response to inflammatory signals by multiple cytokines, Interferon (INF)-γ being the most potent ,27,28.PD-L1/PD-1 interaction in the tumor microenvironment (TME) also promotes T cell dysfunction, exhaustion, apoptosis, neutralization, and production of Interleukin (IL)-10 creating a state of resistance from Cytotoxic T cell (CD8+) mediated tumor cell destruction. All these mechanisms are crucial to hijack the immune system and play a vital role in tumor development, progression metastasis, and survival by escaping tumor immune surveillance ,28. HencThe first ICI to be tested and approved for the treatment of cancer patients was the anti-CTLA-4 blocking antibody—Ipilimumab ,31,32. CT-cell activation requires engagement between the T-cell receptor and the Major Histocompatibility Complex (MHC), but also between co-stimulatory receptors on the surface of the T cell with co-stimulatory ligands expressed by Antigen Presenting Cells (APC). CD28 and CTLA-4 are two important T-cell co-stimulatory receptors that have antagonist mechanisms of action . CD28 isIn summary, the major function of the anti-CTLA-4 is to allow T-cell activation, proliferation, and migration to the tumor tissue mediating the death of tumor cells ,28,35,36Ipilimumab was first approved in 2011 for the treatment of unresectable or metastatic melanoma as it showed an improvement in OS with manageable toxicities. Currently, it is approved in monotherapy or combination with nivolumab, an anti-PD-1 antibody, in multiple settings  or Microsatellite Instability-High (MSI-H), and others) ,39. New PD-1, also known as CD279, is homologous to the CD28 family of protein receptors. PD-1 is predominantly expressed on memory T cells in peripheral tissues after activation by TCR/antigen-loaded MHC and CD28/B7 interactions. It is also vastly expressed on regulatory T cells (TReg) leading to inhibition of immune responses by expression of the forkhead transcription factor FOXP3, absence of expression of effector cytokines (such as IFN-γ), and production of inhibitory cytokines . Less commonly, PD-1 can also be expressed in B cells, activated monocytes, dendritic cells, and Natural Killer (NK) cells .PD-L1, also known as CD274 or B7-H1, and PD-L2, also known as CD273 or B7-DC, are two ligands for PD-1 and both members of the B7 family . PD-L1 iActivation of the PD-1/PD-L1 signaling axis negatively regulates T cell-mediated immune responses by largely reducing cytokine production, namely IFN-γ, Tumor Necrosis Factor (TNF)-α, and IL-2 production. This cascade also has effects on cell differentiation and survival, directly by inhibiting early activation events (positively regulated by CD28) and indirectly through IL-2 ,28. PD-1The inhibition of PD-1/PD-L1 bound by anti-PD-L1 or anti-PD-1 antibodies reverses T-cell suppression and enhances endogenous anti-tumor immunity ,19,44,45The first anti-PD-1 approved was nivolumab in 2014. During the following 4 years, several other inhibitors of the PD-1 receptor or its ligands, PD-L1 and PD-L2, were approved, with pembrolizumab, atezolizumab, durvalumab, and avelumab showing significant enhancement in OS and Progression Free Survival (PFS) in several cancer types.Currently, the inhibitors of PD-1/PD-L1 either in monotherapy or combined with chemotherapy are approved in stage IV NSCLC as the first-line setting providing long-lasting responses and survival ,47. FurtLately, several clinical trials, to evaluate the safety and feasibility of neoadjuvant ICIs in NSCLC have been developed. Their results propose that ICIs could be better tolerated than standard neoadjuvant chemotherapy and more effective in reducing cancer recurrence and metastasis ,6,8,51. phase III trial, which enrolled 773 patients with stage IB to IIIA resectable NSCLC to receive nivolumab plus platinum-based chemotherapy or platinum-based chemotherapy alone, followed by resection. The median Event-Free Survival (EFS) was 31.6 months with nivolumab plus chemotherapy and 20.8 months with chemotherapy alone. The percentage of patients with a pCR was 24.0% and 2.2%, respectively and irrespective of PD-L1 expression. Grade 3 or 4 treatment-related adverse events (AEs) occurred in 33.5% of the patients in the nivolumab-plus-chemotherapy group and 36.9% of those in the chemotherapy-alone group. Moreover, 83% of patients who received nivolumab underwent surgery and achieved a complete surgical resection (R0). Surgery-related and treatment-related AEs were similar in both arms. In conclusion, this study demonstrated that neoadjuvant chemo-immunotherapy does not affect the feasibility of surgery and increases pCR [CheckMate 816 is a phase II trials are ongoing to evaluate neoadjuvant ICI in NSCLC:Several NADIM trial is an open-label, multicenter, single-arm with patients with surgically resectable stage IIIA NSCLC. These patients received neoadjuvant treatment with paclitaxel plus carboplatin plus nivolumab for three cycles before surgical resection, followed by adjuvant nivolumab monotherapy for 1 year. At 24 months, progression-free survival (PFS) was 77,1%, and 30% had toxic events ≥ grade 3 [The  grade 3 . PRINCEPS trial enrolled 30 patients with resectable clinical stage IA-IIIA NSCLC to be treated with one cycle of induction atezolizumab. None of the patients had delayed surgery and all had complete resection (R0). MPR was not observed, however, this could be explained by the short delay between the infusion of atezolizumab and surgery (between 21 and 28 days). Once again, this trial proved the safety and feasibility of neoadjuvant immunotherapy [The otherapy .NCT02716038, included 30 patients with stage IB-IIIA NSCLC treated with four cycles of atezolizumab plus carboplatin plus nab-paclitaxel in a neoadjuvant setting. The primary endpoint was MPR and it was achieved in 57% of patients [The patients .NEOMUN trial included 30 patients with NSCLC stage II/IIIA suitable for curative intent surgery. The patients were treated with two cycles of pembrolizumab, followed by tumor resection. MPR was observed in 27% of the patients with 33% of grade 2-3 related adverse events. In conclusion, neoadjuvant pembrolizumab resulted in a feasible and safe treatment [The reatment .NA_00092076, 21 patients with untreated, surgically resectable early (stage I-IIIA) NSCLC were treated with two preoperative doses of nivolumab. An MPR was observed in 45% of patients, irrespectively of PD-L1 expression [In pression .TOP 1501 enrolled 35 patients with untreated clinical stage IB to IIIA NSCLC, treated with two cycles of pembrolizumab  NSCLC. These patients were treated with three cycles of cisplatin plus docetaxel followed by two doses of durvalumab, surgery, and durvalumab maintenance for 1 year. A total of 55 patients were submitted to surgery, with 62% MPR, 18% pCR, and an EFS of 73% at 1 year [t 1 year .NeoTAP01, a multi-center clinical trial, included 33 patients with stage IIIA or T3-4N2 IIIB NSCLC considered surgically resectable. Patients received three cycles of neoadjuvant treatment with toripalimab plus carboplatin, and pemetrexed (for adenocarcinoma) or nab-paclitaxel (for other subtypes). Surgical resection was performed 4-5 weeks later. MPC was achieved in 60.6% of patients with 45.5% with pCR. Toxicities were manageable [The nageable . LCMC3 trial included a single-arm study to investigate the efficacy and safety of atezolizumab monotherapy, as neoadjuvant therapy, in patients with resectable (IB- selected IIIB) NSCLC patients. A total of 181 patients were enrolled, but only 143 were included in the primary efficacy analysis, 20% presented MPR and 6%pCR [nd 6%pCR . Some other promising ongoing trials with multimodal or new therapeutic combinations approach are being developed  ,60,61. TNormal cells can protect themselves against the harmful effects of Deoxyribonucleic Acid (DNA) damage and keep the integrity of the genome. This genetic stability is maintained through several cellular mechanisms such as regulation of DNA damage signaling, recruiting effector proteins for DNA repair, chromatin remodeling, transcription, and stabilization of replication forks. The actual impact of DNA-damage repair (DDR) gene alterations remain unsatisfactorily explored in oncology, and research concerning its role in the genesis and progression of cancer, and potential therapeutic targets is ongoing, including in lung cancer ,64,65,66Smoking is a major risk factor in lung cancer, it induces DNA damage, and promotes the activation of several repair mechanisms that might offer a rationale for targeting DDR defects in selected lung cancer patients. Even though smoking-related lung cancer is related to a higher tumor mutational burden (TMB), it remains uncertain if specific DDR gene alterations are more common in lung cancer that arises in smokers ,67. Double-strand DNA breaks (DSB) constitute the most severe kind of DNA damage, as they disrupt both DNA chains, leading to mutations or chromosomal reorganizations, increasing the oncogenic risk, and ultimately causing cell death. Homologous recombination Repair (HRR) represents a crucial mechanism for DSB reparation . HRR is These findings explain, at least partially, the reason why alterations in DDR genes are identified in 1/3 of malignancies and raise several questions regarding the role of such deficiencies in lung cancer ,66,68. SSingle-strand DNA breaks (SSB) represent another type of DNA damage. SSBs are fixed by three mechanisms: base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). The vital enzymes to effectively accomplish these processes are from the Poly Adenosine Diphosphate (ADP) Ribose Polymerase (PARP) family. This family includes a group of 17 proteins, of which PARP1-3 are delegated to repair DNA breaks through BER that supply BRCA inefficiency. PARP-1 is the most abundant member of ADP-ribosyl transferases of the PARP family and is responsible for approximately 80–90% of the PARylation activity in cells ,65,68,73BRCA1 and BRCA2 mutations [Hence, BRCA mutant cells are HRR inefficient, so if PARP is blocked they are more prone to accumulate SSBs and produce potential DSBs, inducing genomic instability and culminating in cell death, the so-called “synthetic lethality” ,68. Thisutations ,74. Moreutations ,69,73. Iutations ,69,73,74One of the most important mechanisms of action of PARPi occurs by entrapment of the PARP-1 protein at the replication fork, blocking PARylation reactions (such as transcription and/or translation) and auto-PARylation, increasing PARP1 eagerness for DNA after allosteric changes in its structure. Resulting in a break in the progression of the replication fork, and conducing to a cytotoxic effect, as unrepaired SSBs convert into DSBs, leading to cellular death ,68,69. POlaparib was the first approved PARPi, in 2014, as a maintenance treatment for platinum-sensitive, recurrent serous ovarian cancer patients with a BRCA1/2 mutation who had received at least two previous lines of chemotherapy. This treatment resulted in a noteworthy augmentation in PFS despite not having a significant impact on OS ,77. It wFew trials including PARPi in monotherapy were conducted in NSCLC. Some of the most relevant included platinum-sensitive NSCLC patients, however, they did not show improved outcomes ,84.phase II trials. The PIN trial, a randomized-control trial, included 70 platinum-sensitive NSCLC patients, irrespective of BRCA mutational status, to receive either olaparib or a placebo. Patients that received olaparib had a longer PFS , however, it was not statistically significant [Some of the results already available of the use of PARPi in NCLSC are S1900A, Lung-MAP sub-study, NSCLC patients had a high genomic loss of heterozygosity or BRCA1/2 mutation, progression on platinum-based or anti-PD-(L)1 therapy, and were treated with rucaparib in monotherapy. This study was precociously closed after an interim analysis demonstrated futility [In futility . Hence, the presence of mutations and changes in the expression of crucial DDR genes in lung cancer, notwithstanding having a strong rationale for the use of PARPi, it is recognized that this therapy in monotherapy might not be sufficient to correctly address this disease. However, associations with other therapies, such as immunotherapy, might grant better outcomes ,72,84.Several trials, in different phases, showed benefits in efficacy and safety of PARPi use in monotherapy or association with chemotherapy in neoadjuvant settings in breast cancer, some of which are: ,88,89,90Phase I trials: NCT03329937, which evaluated neoadjuvant niraparib antitumor activity and safety in 21 patients with localized HER2-negative, BRCA-mutated breast cancer. After two cycles, 90.5% had tumor response (≥30% reduction from baseline) by magnetic resonance imaging (MRI). After 2–6 cycles, 40.0% of the patients had pCR; no new safety signals were identified [NCT03499353 trial enrolled 20 patients with breast cancer stage I to III, HER2 negative, BRCA mutated. They were treated with talazoparib for 6 months followed by definitive surgery. Here, 63% had MPR with 53% of the patients having pCR, with manageable toxicity [entified . The NCTtoxicity .Phase II trials: the PETREMAC trial, included 222 patients with stage II/III breast cancer, that were stratified to eight different neoadjuvant treatment regimens based on estrogen and progesterone receptors, HER2 expression as well as TP53 mutational status. The 32 patients with TNBC, irrespectively of BRCA and TP53 mutational status, received olaparib monotherapy for up to 10 weeks, followed by neoadjuvant chemotherapy. Based on combined clinical and MRI evaluation, olaparib treatment yielded 1 clinical complete response and 17 partial responses (ORR of 56.3%). Response to olaparib occurred independent of tumor size. Even in the 27 patients not harboring BRCA 1/2 or PALB mutations, the ORR was 51.9%. Olaparib was well tolerated, with only one patient experiencing grade >2 toxicity requiring a dose reduction [GeparOla trial assessed the efficacy of paclitaxel plus olaparib in comparison to paclitaxel plus carboplatin followed by epirubicin and cyclophosphamide, in neoadjuvant treatment in HER-negative patients with early breast cancer with HRD. A total of 107 patients were randomized, with 69 patients to the arm of Olaparib plus paclitaxel. The pCR rate in the arm with olaparib was 55.1% versus 48.6% in the chemotherapy-only arm. Significantly fewer patients in the olaparib arm experienced grade 3-4 hematologic toxicities 46.4% versus 78.4% (p = 0.002) [the I-SPY2 trial, a combination of durvalumab and olaparib added to standard paclitaxel neoadjuvant chemotherapy  was investigated in stage II-III HER2-negative breast cancer. Seventy-three participants were randomized to the intervention arm and 299 to standard-of-care (paclitaxel) control. DOP increased pCR in all breast cancer subtypes . In the DOP arm, 12.3% of patients had immune-related grade 3 AEs (irAEs) versus 1.3% in control [eduction . The Gep= 0.002) . In the  control . phase III BrighTNess trial, candidates for potentially curative surgery patients with formerly untreated stage II-III TNBC were enrolled. They were randomly assigned (2:1:1) to receive one of three regimens: paclitaxel plus carboplatin plus veliparib; paclitaxel plus carboplatin; or paclitaxel; followed by four cycles of doxorubicin and cyclophosphamide in all patients. A total of 634 patients were randomly assigned: 316 to paclitaxel plus carboplatin plus veliparib, 160 to paclitaxel plus carboplatin, and 158 to paclitaxel alone. pCR was reached in 58% of patients in the paclitaxel plus carboplatin group, 53% of patients in paclitaxel, carboplatin plus veliparib, and 31% in paclitaxel alone. Serious AEs, (≥grade 3) were more common in patients receiving carboplatin, however, veliparib did not significantly augment toxicity. The investigators concluded, that in this study, the addition of carboplatin to paclitaxel improved pCR, however, the addition of veliparib to that same chemotherapy protocol did not [In the  did not .Overall, these data sustain the efficacy of PARPi in a neoadjuvant setting, in monotherapy, or in association with chemotherapy. In the BrightTNess trial results were not affirmative, however there is a potential for bias interference, such as population number asymmetry. Several trials in the neoadjuvant setting in different contexts, such as breast, prostate, and ovarian, with variated associations  are ongoing ,94,95,96A vital mechanism underlying cancer immune evasion is the expression of inhibitory ligands on the surface of cancerous cells, PD-L1 being the most well-known. Historically, PD-L1 has been identified as the first biomarker for ICIs response, despite that it is an imperfect predictor of ICI response. The serine/threonine protein Glycogen Synthase Kinase 3-beta (GSK3β) is an enzyme that controls glycogen metabolism, interacts with PD-L1, and modulates its expression by inducing proteasome degradation of PD-L1. PARPi has been associated with an increase in PD-L1 expression mainly due to the inactivation of GSK3β, in a dose-dependent manner, suppressing T-cell activation and increasing tumor cell killing ,97.Another pathway through which PARPi upregulates PD-L1 is with ATM-ATR-Checkpoint kinase 1 (CHEK1). ATM acts as a kinase sensor for DSBs, after ATM is activated a switch in a signal kinase from ATM to ATR occurs. Finally, the ATM-to-ATR switch activates CHEK1 which further leads to Janus Kinase/Signal Transducer and Activator of Transcription proteins (JAK/STAT) signaling activation and upregulation of PD-L1 expression ,98.H1 immune response, leading to extrinsic tumor suppression, due to infiltration of suppressive immune cells, like Myeloid-Derived Suppressor Cells (MDSCs) or Tumor-Associated Macrophages (TAMs), leading to additional DNA damage via free radical release. These mechanisms lead to chronic inflammation, immunosuppression, and cancer progression. PARP regulates T-cell function, maturation, and differentiation, but also DCs recruitment and functioning, macrophage polarization, and increased MDSCs recruitment to TME. PARPis may have the potential to shift from this chronic inflammatory status to a more immune-responsive TME through extensive effects on cells involved in innate and adaptive immune response and soluble factors, resulting in activation of immunosuppressive pathways thus offering targetable immunological vulnerabilities [DNA damage and deficient mechanisms of DNA repair alter the intrinsic immunogenicity, through modulation of surface phenotype and intracellular pathways, but also by modifying the extrinsic immunogenicity of the TME, which plays a major role in response to therapies ,97. TumoAnother mechanism of interaction between PARPis and the immune system is through the pathway of STimulator of INterferon Genes (STING), a system involved in the production of IFN-γ and pro-inflammatory cytokines. Genomic instability in tumor cells, either induced by PARPi or innate mutations, leads to the accretion of incompletely repaired DNA, producing tumor-derived double-strand DNA (dsDNA) in the cytoplasm. The dsDNA can be detected by cytosolic DNA sensor cyclic GMP-AMP Synthase (cGAS) that leads to activation of the STING signaling pathway. STING stimulates phosphorylation and nuclear translocation of IFN I transcriptional regulatory factors TANK- binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3), it also activates NF-κB pathway which acts with IRF3. IFN I upregulation promotes systemic immune response and regulates multiple components in anticancer immunity, especially T cells, NK cells, and DCs. IFN also stimulates the JAK/STAT pathway which leads to the expression of IFN-related genes. STING upregulation also leads to PD-L1 expression ,97,98,99In many solid tumors, a mutation in genes concerning DNA repair, either innate or acquired, enhances TMB and neo-antigens production, and this has been strongly correlated with ICI responses, even though the optimal TMB cut-off remains unclear across tumor types. Furthermore, TMB further increases neo-antigens production and TILs due to a greater expression of genes involved in immune response, such as the TCR signaling, IFN-γ, and TNF receptors. PARPi induces DNA damage and results in DNA fragment accumulation within the cytoplasm. Thus, resulting in a higher expression of neoantigens exposed in the cell surface, increasing immune response activation and TMB improving immunogenicity, and therefore a better potential response for ICI ,97,99.In summary, PARPi leads to the amplification of STING signaling; upregulation of interferons and chemoattractants; the repertoire of tumor antigens; TMB; T cell activation and recruitment; and the expression of immune blockade targets, like PD-L1 ,68,69. TThus, a therapeutical combination of PARPi and other agents, to improve its effects and overcome resistances, is being explored in many tumor sets. Initial studies combined PARPi with chemotherapy, radiotherapy, TKIs, or other regimens. However, current approaches are based on PARPi combination with immunotherapy, in different types of tumors, with very promising results and evidence of synergy ,99,103. TOPACIO trial, published in June 2018. This phase II trial combines pembrolizumab and niraparib in recurrent ovarian and advanced TNBC. In the ovarian cancer group the ORR was 18% with a Disease Control Rate (DCR) of 65%, nearly one-third of the patients were platinum-resistant and these presented an ORR of 25%. The TNBC patients had an ORR of 21% and a DCR of 49% [One of the first trials to show the benefit of the association between PARPi and ICI in solid tumors was the R of 49% ,105. phase II basket MEDIOLA trial, published in October 2018, included a subgroup with SCLC, the patients were treated with a combination of durvalumab (anti-PD-L1) and Olaparib. From the available data results, patients with platinum-sensitive relapsed ovarian cancer with BRCA mutation showed an ORR of 63%, DCR at 12 weeks of 81%, and median PFS (mPFS) of 11.1 months in the association arm. In patients with ovarian cancer BRCA wild-type, the ORR was 31.3%, DCR 28.1%, and mPFS 5.5 months. The subgroup with relapsed gastric cancer showed an ORR of 10% and a DCR of 26%. In patients with SCLC, the DCR was 29% [The  was 29% ,107. JASPER phase II trial, published in January 2022, the association of pembrolizumab and niraparib was studied in patients with advanced-stage NSCLC. The patients were stratified according to the expression of PD-L1 Tumor Proportion Score (TPS) ≥50% (cohort 1) or 1–49% (cohort 2). In cohort 1, ORR was 56.3%, whereas in cohort 2, ORR was 20.0%. In cohorts 1 and 2, the median Duration of Response (mDoR) was 19.7 months and 9.4 months, mPFS 8.4 months and 4.2 months, and the mOS was NR (non-reached) and 7.7 months, respectively. Grade ≥3 AEs occurred in 88.2% and 85.7% of patients in cohorts 1 and 2, respectively. Safety was consistent with the known profiles of these agents in monotherapy [In the otherapy .phase Ib/II basket nonrandomized controlled trial—JAVELIN PARP Medley—patients with advanced solid tumors, including NSCLC, metastatic Breast Cancer (mBC), ovarian, were treated with an association of talazoparib plus Avelumab. In NSCLC the ORR was 16.7%, mDoR of 17.5 months, in the subgroup with DNA damage response (DDR)-positive ORR was 20% and mDoR NR. This association was generally well tolerated and no new safety concerns were identified [In the most recent study, published in November 2022, a entified .Several trials are testing the efficacy of ICI and PARPi combination in neoadjuvant setting in different tumor locations, mainly in breast cancer with BRCA mutations. However, so far, no results from these trials are available and also there are no trials including NSCLC ongoing ,114,115.In the past, strategies to improve immunotherapy responses, and subsequently oncologic outcomes, were based on biomarker selection, like PD-L1 expression. However, this approach only benefits a specific set of patients. Therefore, in other to improve immunotherapy efficacy, overcome resistances and extend this potential benefit to other populations, studies have been focusing on strategies that combine different treatment modalities, such as immunotherapy with chemotherapy, radiotherapy, TKI, or other therapy combinations.At present, one of the main foci of investigation has been the association of ICIs with DNA-damaging agents, such as PARPi. The rationale for the additive activity of both pharmacologic classes, described in this paper, is based on mechanisms of action already recognized and supported by clinical evidence. Hence, considering that immunotherapy only provides help for some patients, and that early-stage NSCLC still has a high risk of relapse and progression despite the current standard of care treatment (surgery ± chemotherapy ± radiotherapy). Development of clinical trials to access the efficacy and safety of neoadjuvant combined therapy with ICI and PARPi in patients with resectable early-stage NSCLC is a priority. These data will allow us to study newer, and possibly more effective, treatment strategies that might improve the oncologic outcomes of these patients.


Priestia megaterium (previously known as Bacillus megaterium) strain BP-R2, was isolated from the surface-sterilized root tissues of the salt marsh halophyte Bolboschoenus planiculmis. The bacteria strain BP-R2 showed high tolerance to different sodium chloride (NaCl) concentrations and produced the auxin plant hormone, indole acetic acid (IAA), under various tested growth conditions. Inoculation of Arabidopsis and pak choi (Brassica rapa L. R. Chinensis Group) plants with the strain BP-R2 greatly enhanced different growth parameters of the host plants under normal and salt and drought stress conditions compared to that of the mock-inoculated plants. Furthermore, the hydrogen peroxide (H2O2) content, electrolyte leakage (EL), and malondialdehyde (MDA) concentration accumulated less in the BP-R2-inoculated plants than in the mock-inoculated control plants under salt and drought stresses. In summary, the plant endophytic bacterium strain BP-R2 increased host plant growth and stress tolerance to salt and drought conditions.Global warming and climate change have contributed to the rise of weather extremes. Severe drought and soil salinization increase because of rising temperatures. Economically important crop production and plant growth and development are hindered when facing various abiotic stresses. Plant endophytic bacteria live inside host plants without causing visible harm and can be isolated from surface-sterilized plant tissues. Using plant endophytic bacteria to stimulate plant growth and increase environmental stress tolerance has become an alternative approach besides using the traditional breeding and genetically modifying approaches to select or create new crop types resistant to different environmental stresses. The plant endophytic bacterium,  Unlike animals, plants are stationary organisms that cannot change their growth environments and they constantly encounter different biotic and abiotic stresses, including salinity, drought, and extreme temperatures. Soil salinity and water deficiency are two major limiting factors for crop production worldwide that may continuously intensify due to climate change and global warming. Most global fresh water is currently used for agricultural practice and crop production. Extensive and improper irrigation practices also lead to salt accumulation in the soil. It is estimated that around 20 to 50% of irrigated lands are seriously affected by salinity ,2,3. FurAzoarcus, Bacillus, Burkholderia, Enterobacter, Herbaspirillum, Microbacterium, Micrococcus, Pantoea, Pseudomonas, Serratia, Streptomyces, and Stenotrophomonas, and the two predominant genera consist of Bacillus and Pseudomonas [Plant endophytic bacteria live within host plants during different stages of their life cycles without causing infection and harmful effects to the host plants ,13. Thesudomonas ,14. Someudomonas ,17.After successful plant colonization, the beneficial endophytic bacteria facilitate plant growth and provide protection through various direct and indirect mechanisms, which include atmospheric nitrogen fixation, macronutrient  solubilization; plant hormone, siderophore, hydrogen cyanide, and ammonia synthesis; and plant hormone level and response modulation as direct mechanisms ,13,14. TSalicornia brachiate [Cicer arietinum [Haloxylon ammodendron [Nypa fruticans [Salicornia bigelovii [Armoracia rusticana [Seidlitzia rosmarinus [Calendula officinalis [Halocnemum strobilaceum [Haloxylon aphyllum [Tetragonia tetragonioides [Bolboschoenus planiculmis. The Bolboschoenus planiculmis plant is a wetland sedge that has great ecological and agricultural values. B. planiculmis plants are distributed towards the westernmost regions of Spain, easternmost areas in Japan, southernmost areas towards New Guinea, and northernmost regions towards Russia [Bolboschoenus are distributed in a wide range of habitats, including coastal and inland salt marshes, littoral zones of freshwater bodies, and temporarily flooded arable land [B. planiculmis species is a perennial plant that has a highly branched underground rhizome with spherical or elongated tubers that confer a competitive ability to reproduce quickly over other plants [B. planiculmis had been tested in previous ecological studies [B. planiculmis plants develop a circular colony that scatters through the coastal salt marsh in Taiwan. The B. planiculmis plant has become an endangered species in Taiwan due to over exploitation of the coastal land. With its high fitness to abiotic stress, such as drought and salinity [B. planiculmis species may serve as a good candidate host plant to identify beneficial plant endophytes.When plants grow in severe salinity and drought environments, the presence of endophytic bacteria in these plants may help them to survive and grow in these conditions. Several studies have shown the beneficial effects of endophytic bacteria on their host plants, such as the rachiate  plant, Crietinum , Haloxylodendron , Nypa frruticans , Salicorigelovii , Armoracusticana , Seidlitsmarinus , Calenduicinalis , Halocnebilaceum , Haloxylaphyllum , Tetragoonioides , and oths Russia . Plants ble land ,34. The r plants . Althoug studies ,34,36, isalinity , the B. B. planiculmis, as a host plant and successfully identified a beneficial plant endophytic bacterium, strain BP-R2, in this study. We have observed its ability to tolerate different concentrations of sodium chloride (NaCl). Relatively high amounts of the auxin plant hormone, indole acetic acid (IAA), were produced by the strain BP-R2 under various growth conditions. Furthermore, the inoculation of Arabidopsis or pak choi (Brassica rapa L. R. Chinensis Group) plants with the strain BP-R2 significantly improved host plant growth under salt and drought stress conditions. This study effectively demonstrated that the plant endophytic bacterium, strain BP-R2, has the potential to be used as a bioinoculant in agricultural practices to enhance plant growth and salt and drought tolerance. In addition, this was the first study to show that a beneficial plant endophytic bacterium can be isolated from the B. planiculmis plant, the endangered plant indigenous to Taiwan and its possible ecological functions of this strain to help host plants survive in the harsh living environment.In order to identify beneficial bacteria that may promote plant growth and increase tolerance to salt stress, we have used the salt marsh halophyte, Bolboschoenus planiculmis plant samples were collected from the estuary soil of Dajia River at Taichung, Taiwan, located in a subtropical climate region with around 24 °C in annual average temperature. The soil pH values of the collected site ranged from 6.2 to 8.3, and the electrical conductivity of the soil solutions were about 0.6 to 12.0 mmhos/cm. The surface-sterilized root tissues were ground in sterilized mortars and the homogenates were serially diluted to spread on the Luria Broth (LB) agar medium  at 30 °C. The bacteria identity was determined by sequencing and phylogenetic analysis of the 16S rRNA gene. The 16S rRNA gene amplification was obtained with colony PCR reactions using primers E8F (5′-AGAGTTTGATCATGGCTCAG-3′) and U1510R (5′-CGGTTACCTTGTTACGACTT-3′) [Bolboschoenus planiculmis plant were deposited in the GenBank database under accession number OP209759.The endophytic bacteria were isolated and characterized according to the protocols of Ho et al. (2015)  and HwanACTT-3′) . The 16SBolboschoenus planiculmis plant. For growth tests of strain BP-R2 in various conditions, bacteria were grown in LB media at 20 °C, 25 °C, 30 °C, 37 °C, or 45 °C; or in LB media with a pH of 4.0, 5.7, 6.0, 7.5, or 9.0; or in LB media with 0.5%, 1.0%, 2.0%, or 3.0% sodium chloride (NaCl); or in Agrobacterium minimal essential (AB-MES) media [600 to determine the biomass and were then grown for 48 or 72 h. Three biological replicates were used for each growth measurement.The endophytic bacteria strain BP-R2 was isolated from the roots of the S) media  with 2% The fatty acid composition of strain BP-R2 was determined using the MIDI Sherlock Microbial Identification System  according to the manufacturer’s instructions and the protocol of Hwang et al. (2013) . The bacThe Biolog tests  were used to determine the utilization patterns of 95 carbon substrates of the isolated bacteria according to the manufacturer’s instructions and the protocol of Hwang et al. (2013) . Bacteri530) and compared with a standard curve of 0–100 µg/mL IAA.IAA production was analyzed by the Salkowski method as described by Hwang et al. (2021)  with minArabidopsis thaliana (ecotype Columbia) and pak choi (Brassica rapa L. R. Chinensis Group) grown in pots were inoculated with bacteria strain BP-R2. The bacteria cultures grown in LB media at 30 °C to an approximate OD600 value of 0.6–0.8 were used for the inoculations. After inoculation, plant seedlings were continually grown in soil at green houses for further salt and drought stress treatments. For salt stress treatments, the BP-R2-inoculated Arabidopsis and pak choi plants were treated with 250 mM NaCl and 200 mM NaCl for 5 days, respectively. The negative control plants were treated with distilled water instead of NaCl. Subsequently, the NaCl-treated plants were treated with distilled water and recovered for 3 days. For drought stress treatments, both BP-R2-inoculated Arabidopsis and pak choi plants were treated with dehydration for 10 and 5 days by withholding water, respectively. The plants were subsequently re-watered for 3 days for recovery. After salt and drought stress treatments, several plant growth parameters, including the fresh weight (FW), dry weight, rosette diameter, number of leaves, length of leaves, and surface area of leaves, were measured in harvested Arabidopsis and pak choi plants as described previously [Arabidopsis or pak choi plants that were not inoculated with strain BP-R2 and not treated with salt or drought stress were used as the negative controls.The bacteria inoculation and re-isolation assays were performed as described by Hwang et al. (2021)  with mineviously . The AraTo verify the endophytic colonization of the inoculated plants with strain BP-R2, surface-sterilized plant tissues were ground with sterile distilled water and plant crude extracts were serially diluted and plated on LB agar to determine the viable bacteria cell numbers. The sequencing and phylogenetic analysis of the 16S rRNA gene was conducted to determine the bacteria identity. Colony PCR reactions were performed with primers E8F (5′-AGAGTTTGATCATGGCTCAG-3′) and U1510R (5′-CGGTTACCTTGTTACGACTT-3′) of the 16S rRNA gene .2O2) concentrations by a colorimetric reaction with xylenol orange (XO) according to Huang and Hwang (2020) [2O2 concentrations were determined by an absorbance measured at 560 nm (A560) by comparison with a standard curve of 0–100 µM H2O2.Leaves from the mock-inoculated control and the bacteria-inoculated, stress-treated plants were collected and ground to determine the hydrogen peroxide (Hg (2020) . H2O2 co520) by comparison with a standard curve of proline.The proline content was determined using the method of Bates et al. (1973)  with minThe EL amounts of the leaf samples were measured according to Gontia-Mishra et al. (2016)  with minw/v) trichloroacetic acid (TCA) containing 0.5% (w/v) thiobarbituric acid (TBA) and was subsequently heated at 95 °C for 30 min, quickly cooled, and finally centrifuged at 10,000× g for 10 min. The MDA concentration was determined by absorbances measured at 440 nm, 532 nm, and 600 nm. The MDA content was measured as μmol/g fresh weight.The MDA concentrations of the leaf samples were measured according to Hodges et al. (1999)  with minArabidopsis or pak choi plants were inoculated with strain BP-R2, and more than 60 individual plants were examined for bacteria inoculation assays. Error bars were calculated with the Microsoft Excel  STDEVP function. The significance test between treatments was based on Duncan tests, with p < 0.05 considered statistically significant.The plant growth measurements were average values from at least three independent bacteria inoculation experiments. At least 20 different seedlings of Bolboschoenus planiculmis. We initially collected ten bacteria isolates from the B. planiculmis plant. Because the bacterial isolate BP-R2 exhibited high levels of tolerance to 0.5%, 5.0%, 7.0%, 9.0%, and 11.0% NaCl when grown on LB medium at 30 °C and this strain was selected for further characterization. To determine the identity of strain BP-R2, we performed fatty acid methyl esters (FAME) analysis and Biolog tests. FAME analysis revealed that strain BP-R2 had large proportions of the branched fatty acid (iso-C15:0 and anteiso-C15:0) and small amounts of strait chain fatty acids , branched fatty acids , and a mono-unsaturated C16:1 ω11 cis fatty acid. The FAME analysis results of strain BP-R2 matched those of the Bacillus spp.Because the endophytic bacteria-associated halophytic plants can help host plants tolerate salinity environments, we isolated beneficial endophytic bacteria candidates from the surface-sterilized root tissues of the salt marsh halophytic plant, Bacillus megaterium. Additionally, strain BP-R2 showed growth in LB medium with a pH of 5.7 and exhibited acid production with mannitol as a carbohydrate substrate, which is consistent with a previous observation of Bacillus megaterium [The utilization of carbonaceous compounds was further determined with Biolog tests. The substrates that could be used for growth under aerobic conditions at 30 °C were dextrin, D-maltose, D-trehalose, gentiobiose, sucrose, D-turanose, stachyose, D-raffinose, D-melibiose, γ-methyl-D-glucoside, N-acetyl-D-glucosamine, α-D-glucose, glycerol, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, lincomycin, D-gluconic acid, D-glucuronic acid, glucuronamide, quinic acid, L-lactic acid, citric acid, α-keto-glutaric acid, L-malic acid, γ-amino butyric acid, acetic acid, lithium chloride, potassium tellurite, and 1% and 4% NaCl. This substrate utilization pattern of strain BP-R2 showed that the similarity (SIM) index value was 0.562, which suggested its classification as gaterium .Priestia megaterium (previously known as Bacillus megaterium) strain ATCC14581 [Priestia megaterium.To further determine the identity of strain BP-R2, we amplified and sequenced its 16S rRNA gene. The DNA sequence of the PCR product was deposited in GenBank (accession no. OP209759). The resulting sequences of the 1048-bp DNA fragments containing the partial 16S rRNA gene from strain BP-R2 showed 99.4% similarity to the same gene of TCC14581 ,48, suggTo determine if the endophytic bacteria strain BP-R2 exhibits plant growth promotion ability, we examined the amount of the plant hormone, IAA, produced by strain BP-R2 under various growth conditions. Several growth parameters, such as temperature, pH, and different carbon and nitrogen sources, can influence the synthesis of IAA in bacteria ,50. In oFurthermore, different carbon and nitrogen sources were used in the AB-MES medium to determine their effects on IAA production. Strain BP-R2 grew best with starch as the carbon source in the minimal medium at 30 °C for 72 h D, whereaArabidopsis thaliana plants were inoculated with strain BP-R2 grown at 30 °C and the endophytic colonization of the plants was confirmed by re-isolating the bacteria from surface-sterilized inoculated plant tissues. The identities of the isolated bacteria were determined by sequencing and phylogenetic analysis of the 16S rRNA gene. Subsequently, various plant growth parameters were examined in Arabidopsis plants inoculated with strain BP-R2 and in mock-inoculated controls. After inoculation, the presence of strain BP-R2 significantly increased the average Arabidopsis plant fresh weight . The H2O2 content increased 3.3-fold in the control plants after salt stress treatments, whereas it only increased 1.5-fold in the BP-R2-inoculated plants under the same stress treatments  from the Brassica Genus were inoculated with strain BP-R2 to examine its plant growth effects and its endophytic colonization was also confirmed by re-isolating the bacteria from inoculated plant tissues. After inoculation with strain BP-R2, the average fresh and dry weights of the aboveground leaves of the pak choi were heavier than the control plants   . Under Nl plants A,C,E,G. eatments A,C,E,G. l plants B,D,F,H. Priestia megaterium strain BP-R2 from the salt marsh halophyte Bolboschoenus planiculmis. The endophytic bacteria grow in the healthy tissues of living host plants and cause no detrimental effects to the hosts. The endophytic bacteria strain BP-R2 produced significant amounts of the phytohormone auxin and may therefore greatly enhance the aerial and belowground parts of the Arabidopsis and pak choi plants during growth and development. Furthermore, the bacteria strain BP-R2 showed tolerances to a wide range of NaCl concentrations and helped Arabidopsis and pak choi plants achieve better growth under salt stress treatments. In addition, inoculation of the Arabidopsis and pak choi plants with strain BP-R2 assisted the plants in growing larger and better after dehydration treatments. These BP-R2-inoculated plants showed relatively lower induced H2O2, EL, MDA, and proline concentrations than the control plants under salt and drought stress treatments, which correlated well with the improved growth and development shown in these BP-R2-inoculated plants. These data demonstrated that inoculation of plants with the endophytic strain BP-R2 may alleviate the oxidative stress damage caused by salt and drought stresses.Plant growth and production are substantially affected by numerous environmental factors. Salinity and drought are two major abiotic stresses encountered by plants. The application of plant endophytic bacteria to increase plant growth and abiotic stress tolerance has become a less time-consuming approach in comparison to traditional breeding and genetic engineering of abiotic stress tolerant crop varieties. Our study successfully isolated the plant endophyte Priestia megaterium strain BP-R2 was originally known as Bacillus megaterium, which is a Gram-positive, mainly aerobic and spore forming bacterium that exists in widely diverse environments from plant host tissues to soil, rice paddies, dried food, seawater, honey, humans, and blood samples [Escherichia coli [Bacillus megaterium has been developed and industrially utilized for more than 50 years [Bacillus/Bacillaceae genomes and Bacillus megaterium became recognized as Priestia megaterium based on its multiple genomic-scale phylogenetic tree analysis results.The endophytic bacteria  samples ,52,53,54hia coli  and was hia coli ,52,53,5450 years ,53,54,5650 years , extensiBacillus species can form spores, which enhance their ability to endure a broad range of stress conditions and allow their use as bacterial inoculants for agriculture and bioremediation practices [Bacillus species, such as Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Bacillus telluris, Bacillus thuringiensis, and Bacillus megaterium [Bacillus megaterium strain CDK25 can synthesize 13.8 μg/mL of IAA, increase vegetative and biological growth parameters, and enrich the mineral compositions of Capsicum annum L. (chili plants) [B. megaterium strain BM18-2, isolated from the hybrid Pennisetum (Pennisetum americanum x P. purpureum Schumach L.), a perennial C4 bunch grass, displayed 3.0 μg/mL of IAA production, and enhanced plant growth and cadmium (Cd) absorption of hybrid Pennisetum plants when grown in Cd contaminated soil [B. megaterium strain STB1, isolated from contaminated estuarine environment soil, contained IAA biosynthetic genes and increased the biomass of tomato plants [Bacillus megaterium strain RmBm31, isolated from the surface-sterilized root nodules of Retama monosperma, also had several genes involved in IAA biosynthesis pathways and significantly improved Arabidopsis seedling growth [The ractices . Furthergaterium ,58,59,60gaterium . The amigaterium . The bac plants) ,63. Anotted soil . Additioo plants . Anotherg growth .Bacillus species, including B. siamensis, B. megaterium, B. subtilis, and B. cereus, showed relatively higher IAA production at temperatures ranging from 25 to 35 °C and at a pH range of 7 to 8 [Stevia rebaudiana showed better IAA yields at a pH range of 6 to 9 and at temperatures of 35 and 37 °C; these bacteria also improved the growth of wheat and mung bean plants [B. megaterium isolate MJHN1, the B. cereus isolate So3II, and the B. subtilis isolate Mt3b [B. megaterium, B. subtilis, B. cereus, and Pantoea agglomerans, revealed their different preferences for carbon or nitrogen sources for IAA production [Our results showed that bacteria strain BP-R2 had greater IAA production at a temperature range of 25 to 37 °C and a pH range of 6 to 9. Consistent with our observations, several f 7 to 8 ,67. Othen plants . Our stuate Mt3b ,69. Seveoduction ,69,70,712O2), superoxide radicals, and hydroxyl radicals, which serves as a signal to trigger a defense response in plants [Arabidopsis and pak choi plants with endophytic bacteria strain BP-R2 significantly decreased salt and drought stress-induced levels of H2O2, MDA, and electrolyte leakage in plants, which correlated well with the improved growth traits of the BP-R2-inoculated plants. In accordance with our observations, several Bacillus species, including B. megaterium, B. thuringiensis, B. amyloliquefaciens, and B. licheniformis, have been demonstrated to increase plant tolerance to salt or drought stress by producing IAA to increase root growth, water and nutrient uptake, compatible solute accumulation, stress-associated enzyme expression, and ROS accumulation reduction [B. megaterium strains, YC4-R4 and TG1-E1, isolated from the rhizospheric soil of a Spartina anglica plant in China, had abilities to promote plant growth and increase plant drought tolerance [B. megaterium, strain STB1, contained IAA biosynthetic genes and promoted tomato growth under normal and salt-stress growth environments [Arabidopsis plants with the B. megaterium strain BOFC15 significantly increased both leaf and root biomass, primary root length, and lateral root density. Furthermore, in the BOFC15-inoculated plants, the cellular ROS levels, ion leakage, and MDA levels were all reduced under drought and polyethylene glycol (PEG)-induced stress conditions [B. megaterium strain FDU301 showed that several oxidative stress responsive genes were upregulated under PEG-induced arid stress conditions [Arabidopsis and pak choi plants. Furthermore, our study has discovered the potential application of the endophytic bacteria strain BP-R2 as a bioinoculant for the agricultural improvement of plant growth and stress tolerance.High salinity and drought impose two major constraints to plant growth and crop yields. Excessive NaCl concentration in soils may decrease water potential and cause physiological drought conditions, which may lead to the inhibition of water and nutrient uptake in plant roots ,5,6. Dron plants ,6. The gn plants ,6. In theduction ,11,13,52olerance ,73. The ronments . Similarnditions . A transnditions . In summ


The putative sequence type 1 (ST1)-specific AlpST-1 virulence gene was also retained among the serotype Ia/ST1 PRGBS isolates. In addition to tet(M) and erm(B), mef(A)-msr(D) elements or the high-level gentamicin resistance gene aac(6′)-aph(2″), which are both rare in PSGBS, were detected among those MDR PRGBS isolates. In the core single-nucleotide polymorphism (SNP) phylogenetic tree, all invasive ST1 PRGBS isolates with serotypes Ia and III were placed together in a clade with a recombination rate of 3.97, which was 36 times higher than the value found for a clade formed by serotype V/ST1 PSGBS isolates derived mostly from human blood. ST1 has been the predominant sequence type among the PRGBS isolates in Japan, and serotypes Ia and III have been very rare among the ST1 PSGBS isolates. Thus, these lineages that mostly consisted of serotypes Ia/ST1 and III/ST1 PRGBS could possibly emerge through recombination within the ST1 populations.This study aimed to investigate genomic traits underlying the antimicrobial resistance and virulence of multidrug-resistant (MDR) group B streptococci with reduced penicillin susceptibility (PRGBS) recovered from elderly patients with bloodstream infections, which remain poorly characterized. The pangenome was found to be open, with the predicted pan- and core genome sizes being 3,531 and 1,694 genes, respectively. Accessory and unique genes were enriched for the Clusters of Orthologous Groups (COG) categories L, Replication, recombination, and repair, and K, Transcription. All MDR PRGBS isolates retained a core virulence gene repertoire , is recognized as the leading cause of neonatal invasive infections. However, an increasing incidence of invasive GBS infections among nonpregnant adults, particularly the elderly and those with underlying diseases, has been observed. There is a trend toward the increasing occurrence of penicillin nonsusceptibility among GBS clinical isolates, from 4.8% in 2008 to 5.8% in 2020 in Japan. Also, in the United States, the frequency of adult invasive GBS isolates suggestive of β-lactam nonsusceptibility increased from 0.7% in 2015 to 1.0% in 2016. In adults, mortality has been significantly higher among patients with bacteremia than among those without bacteremia. Our study revealed that invasive GBS with reduced penicillin susceptibility (PRGBS) isolates harbor major virulence and resistance genes known among GBS, highlighting the need for large population-based genomic surveillance studies to better understand the clinical relevance of invasive PRGBS isolates. Streptococcus agalactiae, or group B Streptococcus (GBS), is recognized as the leading cause of neonatal invasive infections like sepsis and meningitis. However, an increasing incidence of invasive GBS infections in nonpregnant adults, particularly in elderly and immunocompromised individuals with underlying diseases like diabetes mellitus, has been noted in recent years, where skin and soft tissue infections, bacteremia, osteomyelitis, and urinary tract infections are the most common clinical manifestations , and serotypes Ia, Ib, II, III, and V are most frequently associated with invasive infections , 7, 8. I11–15–alp1), Alp2 , Alp3 , Alp4 , alpha C protein (bca), and Rib (rib); pilus island alleles PI-1, PI-2a, and PI-2b; fibrinogen-binding surface proteins A, B, and C ; laminin-binding protein (lmb); C5a peptidase (scpB); C protein β antigen (bac); cell surface protease A (cspA); GBS immunogenic bacterial adhesin (bibA); hypervirulent GBS adhesin (hvgA); and serine-rich repeat glycoproteins (srr1 and srr2) (18–cyl operon), hyaluronidase (hylB), and the CAMP factor pore-forming toxin (cfb), facilitates the bacterial entry and intracellular survival of this organism  family, comprising Alp1 (encoded by nd srr2) , 18–20. GBS isolates with reduced susceptibility to penicillin (PRGBS), which is the first choice for GBS disease therapy and intrapartum chemoprophylaxis, was first reported from Japan in 2008 , 22. Ami21–Very recently, we have reported the population-level serotype replacement of serotype III by serotype Ia among ST1 multidrug-resistant (MDR) PRGBS lineages during a long-term hospital epidemic . Of noteThe total read counts yielded by Illumina sequencing of eight PRGBS isolates with penicillin MICs of 0.25 to 1 μg/mL that were all invasive isolates available to us  ranged fattR and attL direct repeats (TTAAAGGAATTAGCT), harbored predicted virulence genes scpB and lmb, positioned immediately downstream from scpB, which were surrounded by two ISSag2 elements, and cfb (mef(A), encoding macrolide efflux major facilitator superfamily (MFS) transporter Mef(A), and msr(D), encoding the ABC-F-type ribosomal protection protein Msr(D), located just downstream from mef(A). A 5,657-bp fragment containing the mef(A)-msr(D) genes and their upstream and downstream flanking regions was 99.93 to 99.95% identical to the corresponding region in Streptococcus pneumoniae transposons, such as Tn2009 (AF376746), Tn2010 (AB426626), and Tn6822 (MT489699). The presence of a sequence highly similar to this ICE was predicted in the remaining two serotype Ia/ST1 PRGBS strains, SU85 and SU233; the former contained all of the genes described above, but the latter did not contain the mef(A) and msr(D) resistance genes. The T4SS-type ICE was not predicted in the other four PRGBS isolates, serotype III/ST1 strains SF0942 and SF1510, serotype Ib/ST10 strain SF0680, and serotype III/ST464 (CC23) strain MRY-0181422, which harbored the scpB-lmb linkage and cfb. The presence of the Tn916-associated tet(M) was predicted in serotype Ia/ST1 strains SU85, SU97, SU187, and SU233 and serotype Ib/ST10 strain SF0680, while the tet(M)-erm(B) linkage in the Tn916-like Tn3872 element (AM411624) was predicted in serotype III/ST1 strain SF0942.A putative integrative and conjugative element (ICE) with a type IV secretion system (T4SS-type ICEs) with a size of approximately 240 kb, which has not been described to our knowledge, was found in the draft genomes of two serotype Ia/ST1 PRGBS strains, SU97 and SU187 . The ICE and cfb . The ICEr2) of 0.995 and 0.874, respectively . Roary assigned 1,837 genes (52.0%) to the accessory genome, comprising 911 shell genes (25.8%) found in 15 to 95% of genomes and 926 cloud genes (26.2%) found in less than 15% of genomes. The pan- and core genome prediction curves yielded correlation coefficients (ectively . The sizectively . Though ectively .The distribution of the clusters of orthologous groups (COGs) of proteins in core, accessory, and unique genes is shown in pbp2a gene of this strain. Comparative growth curve analysis was performed to investigate the impact of this disrupted PBP2A on the cellular growth of this strain. As shown by the results in Among the six ST1 PRGBS isolates with a penicillin MIC of 0.25 μg/mL , serotypAmino acid substitutions in the QRDRs of the DNA gyrase (GyrA) and topoisomerase IV (ParC) have mainly been associated with fluoroquinolone resistance of GBS. Two serotype Ia/ST1 PRGBS strains, SU85 and SU97, with a levofloxacin MIC of 2 μg/mL  shared SR/M) per branch varied mostly depending on the ST (CC) to which the isolates belonged  isolates mostly derived from the same hospital where the four serotype Ia isolates were obtained, formed an independent clade. The R/M value for the clade was found to be 3.97, which was 36 times higher than the R/M value of 0.11 for a serotype V/ST1 clade that mostly consisted of penicillin-susceptible GBS (PSGBS) isolates from human blood. The CC23 clade that predominantly consisted of serotype Ia and III isolates, including serotype III/ST464 PRGBS strain MRY08-1422, exhibited an R/M value of 0.88. An R/M value of 5.7 was estimated for the clade comprising subclades of CC283 that consisted predominantly of fish isolates, CC10 isolates including serotype Ib/ST10 PRGBS strain SF0680 and rat isolates, and CC7 isolates that consisted predominantly of fish isolates. Camel isolates were divided into two clusters, of which one consisted of seven isolates with an R/M value of 6.34 and the other of two isolates with R/M values of 0.03 to 0.06.The recombination-mutation ratio , ISSag4 for serotype II/ST2 strain CH17, and a Streptococcus phage Javan53 mobile element for serotype V/ST609 strain ILR1005. The absence of lmb and scpB genes in the CC103 and CC67 clades, which mostly consisted of cow isolates, serotype Ib/ST12 rat isolates from the CC10 clade, and a clade of camel isolates, was noted.A whole-genome multilocus sequence typing (wgMLST) tree of the 8 invasive PRGBS isolates and 92 reference strains for which genomes were available in NCBI  and the repertoire of virulence- and resistance-associated genes detected in their genomes are shown in tet(M) (tet(M) gene was also detected in most of the reference isolates of human origin, whereas it was scarce among isolates from fish, camels, and cows. Strains SF0680 and SF0942 had erm(B), and strain SF1510 had tet(O). Three serotype Ia/ST1 PRGBS strains, SU85, SU97, and SU187, had mef(A)-msr(D), which was also detected in only five of the reference isolates of human origin. The aminoglycoside resistance gene aac(6′)-aph(2″) was detected in serotype III/ST464 PRGBS strain MRY08-1422 and one of the reference isolates, strain HU-GS5823. However, a 4.5-kb fragment containing aac(6′)-aph(2″) surrounded by IS256 elements, the nucleotide sequence of which was identical to that of staphylococcal transposon Tn4001, was identified only in strain MRY08-1422 (mre(A) gene was found in all PRGBS/PSGBS isolates analyzed. The mre(A) gene, encoding a flavokinase, was originally reported as a macrolide efflux gene in S. agalactiae  . The tetY08-1422 . The mrealactiae . Howeveralactiae .pezTA toxin-antitoxin genes were identified within a truncated form of a Tn5253-like element. The 1,247-bp fragment containing pezT and pezA that was located upstream from pezT exhibited 97.8% and 93.7% nucleotide sequence identities with those of S. pneumoniae  and S. agalactiae , respectively. These four isolates also harbored CAAX protease and bacteriocin-processing (CPBP) family intramembrane metalloprotease genes in a 1,221-bp fragment that exhibited 99.9% nucleotide sequence identity with that of S. pneumoniae . The BLAST analysis showed a low query coverage of approximately 44% (96% nucleotide sequence identity) for that fragment from S. agalactiae .In addition, manual exploration of the Roary gene_presence_absence.csv file and Microbial Nucleotide BLAST analysis found a few putative virulence genes among four invasive PRGBS isolates of serotype Ia/ST1. Namely, In our previous study, a poor clinical outcome was observed in all four elderly patients who had serotype Ia/ST1 PRGBS isolated from blood cultures , promptiThis first genomic study of invasive PRGBS in Japan confirmed the close genomic relationship of four serotype Ia/ST1 PRGBS isolates of nosocomial origin. Three of these serotype Ia/ST1 strains, SU97, SU187, and SU233, were detected at 1, 14, and 20 months, respectively, after the isolation of SU85 . Our preThe pangenome of GBS has consistently been found to be open, with the pangenome curve showing no evidence of plateauing , 40. TherPSGBS reference isolates mostly derived from the same hospital where the serotype Ia isolates were obtained was separated from a clade of serotype V/ST1 reference isolates mostly derived from human invasive infections. It was found that genetic recombination occurred more frequently within the former clade than in the latter clade. The increasing impact of invasive ST1 GBS infections, predominantly associated with serotype V, in nonpregnant adults has been recognized. Genomic evolution of these serotype V/ST1 isolates by converting to serotypes Ib, II, and IV through large-scale recombinations  phylogenomic tree of 100 genomes showed concordance with their ST (CC) distribution. Notably, a clade comprising our six invasive PRGBS isolates of serotypes Ia and III/ST1 and four ST1 PRGBS/CTBinations  is obserinations . ST1 hasinations , 31, andinations , 47. Theinations , 40, 46.bibA, fbsA/-B/-C, cspA, cfb, hylB, scpB, lmb, and the cyl operon), supporting an invasive ability of our PRGBS isolates comparable to those of other human invasive PSGBS and noninvasive PRGBS isolates. Additionally, the presence of the AlpST-1 virulence gene was confirmed in four serotype Ia/ST1 PRGBS isolates, which may provide an additional benefit for their colonization  21–, 26, 38.tet(M), and the three of those strains that were also resistant to erythromycin harbored mef(A)-msr(D). The acquisition of mef(A)-msr(D) by GBS/PRGBS is rare, but three serotype III/ST17 GBS isolates from the blood of neonates possessing mef(A)-msr(D) were noted in our analysis. The serotype Ib/ST10 strain SF0680 and serotype III/ST1 strain SF0942, which exhibited resistance to erythromycin, clindamycin, and minocycline, had erm(B) and tet(M) genes, while the serotype III/ST1 strain SF1510, which was resistant to minocycline, had the bovine-associated tet(O) gene. Of note, the presence of the aac(6′)-aph(2″) gene, encoding a bifunctional enzyme conferring high-level resistance to gentamicin, was first confirmed in Japan in an invasive PRGBS isolate (serotype III/ST464 strain MRY08-1422) exhibiting high-level aminoglycoside resistance . The aac(6′)-aph(2″) gene is prevalent in enterococci and staphylococci, while it is still uncommon in GBS, representing only 0.27% of 6,340 isolates recovered during 2015 to 2017 through population-based Active Bacterial Core surveillance (ABCs) in the United States (4001 carrying aac(6′)-aph(2″), so a risk may lie in the spread of this resistance gene among GBS/PRGBS as a vector.The eight invasive PRGBS isolates harbored different sets of antimicrobial resistance genes according to their phenotypic resistance profiles. Namely, the four serotype Ia/ST1 PRGBS strains, SU85, SU97, SU187, and SU233, exhibiting resistance to minocycline, harbored d States . This stpezT and pezA were found within a putative Tn5253-like element. Activation of PezT through proteolytic degradation of PezA during environmental stress or bacterial infections would trigger the lysis of bacterial cells, resulting in the release of virulence factors that facilitate the expansion of infectious lesions -msr(D) or aac(6′)-aph(2″) was noted. There is a trend toward increasing occurrence of penicillin nonsusceptibility among GBS isolates from various clinical sources, from 4.8%  in 2008 to 5.8%  in 2020 according to the Japan Nosocomial Infections Surveillance (JANIS) data of the Ministry of Health, Labor and Welfare (https://janis.mhlw.go.jp/english/index.asp). In the U.S. Active Bacterial Core surveillance (ABCs) of invasive GBS isolates from nonpregnant adults, the frequency of isolates with elevated MICs or pbp2x variants suggestive of β-lactam nonsusceptibility increased from 0.7%  in 2015 to 1.0%  in 2016  using a Biowave CO8000 cell density meter . All experiments were performed three times independently.For growth curve experiments, overnight cultures were inoculated 1:100 into 50 mL of THB and the cells were incubated aerobically with agitation at 37°C. Growth was monitored over time by measuring the optical density at 600 nm . Alternatively, broth microdilution panels prepared in-house were used to provide a broader range of antimicrobial concentrations for evaluation of the MICs of ciprofloxacin  and gentamicin  . The susde novo assembly were conducted as described previously (de novo using SPAdes version 3.14.1. Molecular serotypes and STs were confirmed in silico using the genomic sequences according to previously described methods (https://www.ezbiocloud.net/tools/orthoani) (Whole-genome sequencing (WGS) and eviously . Briefly methods . The overthoani) .oriT, relaxase, and type IV coupling protein [T4CP]) and mating-pair formation elements (Tra and VirB4) were detected by ICEfinder (https://bioinfo-mml.sjtu.edu.cn/ICEfinder/index.php). Virulence genes and antimicrobial resistance genes in the putative ICEs, assembled genomes of 8 PRGBS, and 92 reference genomes available from the NCBI, comprising 2 PRGBS and 1 CTBrPSGBS isolate in Japan, 7 PRGBS isolates from other countries, and 82 PSGBS isolates (Table S1), were analyzed using VFAnalyzer from the virulence factors database , the comprehensive antibiotic resistance database , ResFinder 4.1 from the Center for Genomic Epidemiology (http://www.genomicepidemiology.org), and the BLASTn tool. Manual verification of the nucleotide sequences of those predicted virulence genes and resistance genes was performed on WGS data.Putative T4SS-type ICEs carrying genes encoding mobilizable elements , producing output gff3 files that were used for pan- and core genome analyses using Roary  on a Galaxy-based platform. An ML phylogenetic tree based on the core genome alignment was generated with RAxML in Galaxy. The Roary gene_presence_absence.csv file and the core genome tree were visualized using Phandango  was used as the reference. The resulting alignment in XMFA format, which was converted to FASTA format, was then used as the input for Gubbins  to perform recombination analysis and filter SNPs within recombinant regions in the core SNP alignment. The resulting tree and recombination events were visualized using Phandango (A single-nucleotide polymorphism (SNP)-based maximum-likelihood (ML) phylogenomic tree and SNP densities used to predict recombination and horizontal gene transfer events were generated across the genomes of 100 isolates comprising the 8 invasive PRGBS isolates in this study and 92 reference strains by using Gubbins (Table S1). Namely, the core genome SNPs based on whole-genome alignments of the 100 genomes were generated using Parsnp (version 1.5.6) implemented in the Harvest suite . S. agalhandango .http://wgmlstdb.imst.nsysu.edu.tw) (Table S1). The phylogenetic tree was visualized using Interactive Tree of Life (iTOL) version 5 (http://itol.embl.de/).A whole-genome multilocus sequence type (wgMLST) tree of the 100 genomes was constructed based on alleles of all genes in the pangenome using PGAdb-builder (GCA_020111495.1, GCA_020111445.1, GCA_020111535.1, GCA_020111505.1, GCA_020111555.1, GCA_020111435.1, GCA_020111455.1, and GCA_020111565.1 and BioProject accession number PRJNA649379.Genome assemblies for the eight PRGBS strains, SU85, SU97, SU187, SU233, SF0680, SF0942, SF1510, and MRY08-1422, have been deposited in the DDBJ/EMBL/GenBank database under GenBank assembly accession numbers


Meanwhile, the biological functions of SCAF11 in BRCA were further validated with several algorithms, such as Xiantao tool, LinkedOmics, GEPIA2, and TISIDB. These findings indicated that the expression of SCAF11 was significantly correlated with diverse tumor-infiltrating lymphocytes (TILs), including T central memory cell (Tcm), and type 2 T helper cell (Th2), etc. Functional enrichment analysis suggested that co-expression genes of SCAF11 primarily participated in inflammation and immune-related signaling pathways, such as oxidative phosphorylation, antimicrobial humoral response, and immunoglobulin complex. Moreover, SCAF11 expression was positively correlated with several immune checkpoints, including PD-L1, B7H3, and PDCD1LG2. Taken together, this study uncovered that pyroptosis-associated gene signature might be applied as an effective independent predictor in patients with BRCA. The pyroptosis-related gene SCAF11 might play potential roles in the regulation of immune microenvironment in BRCA.Pyroptosis, characterized as an inflammasome-mediated cell death pathway, may be participated in tumorigenesis and progression. However, the underlying molecular function and mechanism of pyroptosis in BRCA remain unclear. In our study, we aimed to develop a prognostic signature in BRCA based on pyroptosis-associated genes. Data was downloaded from TCGA database, and then we screened 760 female BRCA samples and 104 normal breast tissues as the training set. Seven pyroptosis-related genes  were identified as the pyroptosis-related prognostic model for BRCA using LASSO Cox regression. We subsequently tested the prognostic value of pyroptosis-associated gene signature in a validation set, GSE 20685. Time-dependent receiver operating characteristic analysis demonstrated the credible predictive capacity of this pyroptosis-associated gene signature. The area under the curves were 0.806 at 3 years, 0.787 at 5 years, 0.775 at 8 years, and 0.793 at 10 years in the training set, and 0.824 at 5 years, 0.808 at 8 years, and 0.790 at 10 years in the validation set. Furthermore, there are currently few data on SCAF11 regulating pyroptosis. To clarify this issue, we performed integrative bioinformatics and experimental analysis. Knocking down SCAF11 possessed an anti-cancer effect in terms of inhibiting cell viability and suppressing colony-formation in  Breast cancer (BRCA) is the most commonly diagnosed malignant tumor worldwide and the leading cause of cancer-related mortality in women, with an estimated 2.3 million new cases and 685 thousand deaths in 2020 . Risk faPyroptosis is characterized as an inflammasome-mediated programed cell death modality that results in the cleavage of gasdermin D (GSDMD) or gasdermin E (GSDME) and secretion of proinflammatory cytokine IL-1β and IL-18 through pores in cell membrane, subsequently cell swelling, plasma membrane lysis, and eventually, cell death –8. RecenIn this study, BRCA-associated molecular and clinical data were obtained from The Cancer Genome Atlas (TCGA) database. A prognostic risk model for female BRCA was developed based on 33 pyroptosis-related genes, and the prognostic value and clinical characteristics were then evaluated with this risk score model. In addition, we verified this prognostic model with dataset GSE 20685 acquired from Gene Expression Omnibus (GEO) database. The model consisted of seven pyroptosis-related genes, including CASP9, GPX4, IL18, NLRC4, SCAF11, TIRAP, and TNF. As there were few data on SCAF11 regulating pyroptosis, we further investigated the potential roles of SCAF11 in BRCA through experiments and bioinformatics analysis. The expression level of SCAF11 was up-regulated in BRCA, and knockdown of SCAF11 expression possessed an anti-cancer effect in inhibiting cell viability and suppressing of colony-formation. Functional enrichment analysis suggested that co-expression genes of SCAF11 primarily participated in inflammation and immune-associated signaling pathways. Moreover, SCAF11 expression was positively correlated with several immune checkpoints, such as programmed cell death ligand 1 (PD-L1), B7H3, and programmed cell death 1 ligand 2 (PDCD1LG2). In general, this study indicated that pyroptosis-associated gene signature might be an effective predictor in BRCA patients and may pave the way for the development of effective immunotherapy for BRCA.https://portal.gdc.cancer.gov/repository), and we screened 760 female BRCA samples and 104 normal breast tissues for further research as depicted in http://asia.ensembl.org/index.html). GSE 20685, acquired from the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20685), was used as a validation dataset of the prognostic model. We performed the entire data analysis with the R statistical programming language (version 4.0.3).The mRNA expression levels from RNA-Seq and matching clinical data of TCGA-BRCA cohort were obtained from TCGA database (P < 0.20). Eventually, 7 pyroptosis-related genes were selected to establish a prognostic risk model.In this study, we extracted 33 pyroptosis-related genes from prior literature  13–16).–16.13–162. In addition, BT549 and T47D cells were transfected with SCAF11-targeted small interfering RNA (siRNA). The sequences of siRNAs are listed as follows: SiSCAF11-1 5′-CGAACATCCTAGATCTACA-3′; SiSCAF11-2 5′-GCAATCCATTTAACATTCA-3′.Human BRCA cell lines, BT549 and T47D, were cultured in Roswell Park Memorial Institute (RPMI) 1640  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  in a humidified incubator at 37°C with 5% COThe cultured cells were collected and then lysed with IP lysis buffer supplemented with proteinase inhibitors. Equal amounts of total protein (30 μg) were loaded into each lane of 10% SDS–PAGE. Subsequently, proteins were transferred into PVDF membranes and blocked in 5% skimmed milk at RT for 2 h, and then incubated overnight at 4°C with primary antibodies in 5% BSA, followed by HRP-conjugated secondary antibody at RT for 1 h. Housekeep gene GAPDH served as an internal control. Primary antibodies included SCAF11 antibody  and GAPDH . Finally, proteins were visualized using Immobilon Western chemiluminescent reagents .3 cells/well). At day 1, day 2, day 3, day 4, day 5, day 6, cell viability was assessed by CCK-8 assay  reading absorbance at 450 nm using a VICTOR X2 microplate reader  according to the manufacturer's protocols.BT549 and T47D cell lines were transfected with SCAF11-targeted siRNA or control siRNA for 48 h and then seeded in 96-well plates (2 × 103 cells/well). After incubating for about 14 days, cell colonies were counted by staining with 0.3% w/v crystal violet/methanol.BT549 and T47D cells were transfected as previously described and then seeded in 6-well plates (10Least absolute shrinkage and selection operator (LASSO) Cox regression with a 10-fold cross-validation was performed to determine the contributions of pyroptosis-related genes in survival prediction of TCGA-BRCA training cohort . We got https://www.xiantao.love/products) is a comprehensive bioinformatics analysis portal for exploring differential expression, function enrichment analysis, interaction networks, and clinical significance across various cancers. We used Xiantao tool for differential expression of pyroptosis-related genes, gene set enrichment analysis (GSEA), and interactions between BRCA and immune system. Meanwhile, TISIDB . The results showed that 7 pyroptosis-related genes, CASP9, GPX4, IL18, NLRC4, SCAF11, TIRAP and TNF, significantly correlated with prognosis  for each sample was calculated with the “glmnet” R package , including T central memory cell (Tcm), and type 2 T helper cell (Th2) . we alsoWe further explored the correlations between SCAF11 expression and other immune signatures, such as immunoinhibitors, immunostimulators, and major histocompatibility complex (MHC) molecules, using the TISIDB database. As provided in In this study, we established a unique prognostic evaluation model by integrating traditional clinicopathological parameters and risk score based on pyroptosis-related genes in a large-scale BRCA cohort, including TCGA-BRCA as a training dataset and GSE 20685 as a test dataset, and demonstrated its sensitivity and specificity. Furthermore, the relationship between SCAF11 and immune microenvironment indicated that it may be responsible for the poor prognosis for BRCA patients. The nomogram established a link between risk score and clinical outcome. Factors affecting the prognosis of BRCA patients were considered from multiple perspectives, laying a firm foundation for clinical management and treatment.Salmonella typhimurium , a member of caspase family protease, has been identified with apoptotic and non-apoptotic roles, including autophagy regulation , also known as splicing factor, arginine/serine-rich 2-interacting protein (SFRS2IP), is a member of human SR (Ser/Arg-rich) superfamily. Few data are currently available on the regulation of pyroptosis by SCAF11 , 29. In gulation  and endogulation . GPX4  can be found in the article/LC and QY: acquisition of data. YY, ZL, FJ, and QH: analysis and interpretation of data. ZX: conception and design. LO, SW, CF, and YL: data curation. QY and ZX: development of methodology. QY and JP: writing the manuscript and revision of the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by grants from the Changsha Natural Science Foundation (kq2007071), the Natural Science Foundation of Hunan Province (2021JJ30904), the horizontal project (1 43010100), and the Science and Technology Innovation Program of Hunan Province (2021RC3029).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.