Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR /Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host’s immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa. CRISPR /Cas proteins constitute a prokaryotic immune surveillance system in bacteria and archaea to defend against the infection of phages ,2,3. RecBase editing is a new powerful technology that enables the programmable conversion of single nucleosides in the eukaryote and prokaryote genomes . Base edPseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker, primarily infecting commercial cultivars of Actinidia deliciosa and A. chinensis [Psa infects the kiwifruit plant through stomata, stylar, and wounds [Psa-infected plant displays only minor symptoms in summer, while in winter, with humid conditions, Psa colonizes various kiwifruit tissues and rapidly becomes systemic, leading to the death of the host plant [Psa is a very aggressive pathogen to kiwifruit worldwide, we know little about its pathogenic mechanism. The type III secretion system (T3SS) has been shown to be closely associated with the pathogenicity of bacteria [Pseudomonas syringae pv. tomato DC3000, which is used as a model organism for studying plant pathogenicity, has already investigated the function of some effector proteins secreted by the T3SS, such as HopF2Pto and HopAO1Pto [Psa has high homology with the DC3000 effector protein, we can refer to the study of DC3000 effector proteins to explore the pathogenic mechanism of Psa.Bacterial canker severely threatens kiwifruit cultivation worldwide . Pseudomhinensis . Psa infd wounds . Psa-infst plant . Althougbacteria . PseudomopAO1Pto ,20. SincPsa strains is homologous recombination (HR) [Psa is that they are cumbersome. In addition, they are challenging to use for knockout multi-site or high-throughput gene mutation experiments.The currently used method for constructing mutations in ion (HR) ,22,23. Sion (HR) . The RecPsa genome [Psa might depend on NHEJ without donor DNA. The applicability of this method may be limited because it generates large fragment deletions, and deletion sites are uncontrollable [Recently, the genome editing tool CRISPR/Cas9 system has been used to create large fragment deletions  in the a genome . Generala genome . Howeverrollable .Psa. We showed that BE3 achieved accurate and effective base conversion in Psa. We also demonstrated that BE3 could target multi-site base conversion by a single plasmid. Our purpose is to provide a new, simple, and high-throughput tool to investigate the function of Psa genes.Base editing has been demonstrated as a powerful tool with high mutation frequencies in bacteria ,27,28. IThe genomic DNAs of strain PSA.AH.01 (PRJNA923731), isolated from Sichuan Province in China, were sequenced by Oxford Nanopore Platform . A circular chromosome and a circular plasmid were obtained by genome de novo assembly. The length of the chromosome was 6,529,123 bp, and the length of the plasmid was 77,260 bp, for a total of 6,606,383 bp. The GC content of the chromosome was 58.42%, and the plasmid was 56.00%. The assembled data of PSA.AH.01 were subjected to Glimmer, version 3.02, and 6079 protein-coding sequences (CDSs). Furthermore, 65 tRNA genes, 16 rRNA genes, and 43 sRNA genes were identified. Subsequently, PSA.AH.01 was annotated using the Bacterial Virulence Factor Database [Psa, we constructed a dCas9-BE3-fliC plasmid vector that harbors gRNA to target the flagellin (fliC) gene in Psa. The sgRNAs were designed between the 382 bp and 401 bp of the fliC open reading frame to truncate the protein  or CGA (Arg) codons into respective TAG/TAA/TGA premature Stop codons. To test the efficiency of the dCas9-BE3 system in  protein A. The dCost 100% B. Scannidetected C,D. The Psa, we tested dCas9-BE3 at twelve target gene sites with the NGG PAM sequence  in hopAI1 and the 177th amino acid Gln (CAG) in hopI1 were successfully mutated to stop codons (TAG). The sequencing of avrE1 and hopR1 in positive colonies showed that the CAG or CAA (Gln) codons were successfully converted to a TAG or TAA stop codon . The number of plasmid DNAs was approximately 1657 times of library size. All clones were harvested and combined to prepare the plasmid DNAs constituting the sgRNA library. The primers BE3F/R were used to amplify the plasmid DNA library and PCR products were deeply sequenced by Next-generation sequencing (HiSeq 2500). The sequence data showed that 9464 of the 9467 sgRNAs were represented by at least one read (99.97%), and these 9464 sgRNAs covered 5725 genes (99.9%). Moreover, 90% of the sgRNAs had approximately 1721 reads, and 10% of the sgRNAs had less than 460 reads. We randomly selected 365 clones and sequenced them. The results showed that 307 of the 365 clones harbored the correct sgRNA target sequences (84.1%), 55 of the 365 clones had mutated sgRNA target sequences (15.1%), and three clones did not harbor the target sequence (0.8%) . In total, 9467 target sites were identified and located in 5728 genes. The two 25-bp homologous arms derived from the dCas9-BE3 were added to the two ends of the 20-bp guide sequences. Furthermore, 9467 oligonucleotides were synthesized by array-based oligo-pool synthesis. Then the oligonucleotides were amplified, purified, and ligated into the pBmBE3 vector. The recombinant plasmids were transformed into e (0.8%) . Those rPsa mutant strains constructed by BE3 systems, ‘Hongyang’ tissue culture plantlets were incubated with ΔhrcC, ΔhopR1, ΔhopF2, and ΔhopAO2 mutant strains. After 20 days, ‘Hongyang’ plantlets inoculated with Psa3 developed leaf yellowing and browning. The plantlets inoculated with the ΔhrcC mutant (the type III secretion system) appeared healthy and asymptomatic  with the previous studies of dCas9-BE3 and dCas12a-BE3 in Psa. The results showed that the proportions of genes with dCas9-BE3 knockout targets in the Psa genomes were 94.23%, 95.85%, 96.68%, 96.44%, and 96.36%, respectively, with a mean of 95.91%. The proportions of genes with dCas12a-BE3 knockout targets in the Psa genome were 54.07%, 55.35%, 56.92%, 57.57%, and 58.17%, with a mean of 56.42%  PAM, we thought that the dCas9-BE3, which recognized GC-rich (NGG) PAM, was more suitable for gene knockout in Psa genome sequencing, the challenge in the post-genome era is to study the function of the Psa genes systematically. Gene knockout is the most common and effective way to achieve this goal. Generating large-scale mutants is of great value for studying Psa functional genes. Traditional methods for producing large-scale mutations include physical, chemical, and biological methods. The biological methods include transposon insertion and homologous recombination. The transposon insertion method is random and inserts only 47% of chromosomal genes into Psa [E. coli were deleted [Psa genome to a base T, so the CGA/CAA/CAG of the target gene was mutated to a stop codon and finally generated a mutation. It is because of the convenience that it has been successfully applied to eukaryotic and prokaryotic cells. However, the construction of large-scale mutant libraries of Psa is rarely reported. Therefore, we constructed a high-throughput Psa mutant library based on the BE3 system to provide an experimental basis for studying gene function. Since there is an almost one-to-one relationship between sgRNA and target genes in these BE3 mutants, the disease-associated genes can easily identify with observed phenotypes.With the into Psa . The hom deleted . The CRIPto in Pseudomonas syringae pv. tomato DC3000 can bind to the RIN protein of Arabidopsis and not trigger the plant’s ETI response, thereby promoting the growth of DC3000 within the plant [Pto can also interact with and inactivate the Arabidopsis MKK5 protein, reducing the plant’s defense ability [Pto can interact with BAK1, inhibiting the phosphorylation of the BIK1 protein in Arabidopsis and reducing the non-host immunity of plants [Pto in tomato requires its myristoylation site and the catalytic residue of ADP-RT, with the 71st arginine (Arg71) and the 175th aspartic acid (Asp175) of HopF2Pto being particularly important [Pto revealed that it has a protein sequence of 204 amino acids, while HopF2Psa in PSA.AH.01 has 205 amino acids with a homology of 57%. The 72nd amino acid of HopF2Psa is also Arg72, and the 174th amino acid is Asp174, indicating that HopF2Psa may also interact with proteins such as RIN, MKK5, and BAK1 in kiwifruit (The study found that HopF2he plant . HopF2Pt ability . Additiof plants . The virmportant . Furtheriwifruit .Pto is similar to protein tyrosine phosphatases (PTPs) and has PTP activity, which requires a conserved catalytic cysteine residue (Cys378) [Pto can inhibit the HR response of tobacco, but when Cys378 is changed to Ser, this inhibition disappears, highlighting the importance of Cys378 in the role of HopAO1Pto [Pto acts on the tyrosine 836 site of the Arabidopsis EFR protein, reducing its phosphorylation and preventing the downstream immune response [Pseudomonas syringae, with DC3000 possessing only HopAO1 and PSA.AH.01 having only HopAO2. HopAO2 has phosphatase activity and can inhibit early defense responses in tobacco, reduce reactive oxygen species, and reduce callose deposition [Pto in DC3000 is VHC378NGGRGR384T, and the conserved domain of HopAO2Psa in PSA.AH.01 is IHC243GVGQGR249T  . HopAO1PopAO1Pto . The stu8 . HopAOposition . The conposition , the actGQGR249T . Based of DC3000 . Jayaram results . HoweverPsa strain (PSA.AH.01) isolated in China (Sichuan province) from a leaf spot lesion of A. deliciosa cv. Hayward was routinely grown in King’s B medium (KB) at 25 °C. The Escherichia coli strain (Trans T1)  was cultivated at 37 °C in Luria–Bertani (LB) or on LB agar plates. Antibiotics and additives were used at the following final concentrations (μg/mL) unless otherwise noted: Gentamicin (Gm), 50; rifampicin (Rf), 50; theory Isopropyl β-d-1-thiogalactopyranoside .The The genome of PSA.AH.01 was sequenced by the DNBSEQ platform and Nanopore platform . Sequence data were assembled using Falcon, version 0.3.0, and Celera Assembler, version 8.3, and analyzed using Glimmer, version 3.02. Then the protein sequence of the genome was blasted with the VFDB for gene annotation to find the potential virulence factors.PstI restriction sites were removed using the Fast Mutagenesis System . The crRNA-BsaI sequences were synthesized and inserted into the site between BsaI and PstI by T4 DNA Ligase , resulting in pdCas12a-BE  or CGA (Arg) codons were located at 20 bases upstream of the PAM site, then this region would be suitable as a target. For the dCas12a-BE system, finding the TTTN structure in half of the target gene CDS is necessary. If the CAG/CAA (Gln) or CGA (Arg) codons were located at 20 bases downstream of the TTTN structure, this region could be designed as a target site.pBmBE3 or pdCas12a-BE vectors.For a single-site knockout, the target region’s sense and anti-sense strands were synthesized and annealed to form a dimer. For two-site knockout, the ‘Target1-sgRNA-Trc-Target2’ was assembled by overlapping PCR. Then the fragment was ligated into BsaI-digested Psa, we used Python to screen out one or two suitable targets for each gene in the PSA.AH.01 genome. Twenty-five base pairs were added at the 5′ end of the target, which identifies with upstream BsaI in pBmBE3.For the dCas9-BE3 system, the 12-bp seed sequence of the sgRNA should only match with one site in the target genome, so the possibility of off-target editing is very low. If the CAG/CAA (Gln) or CGA (Arg) codons were located at 20 bases upstream of the PAM site, then this region would be suitable as a target. Hence, to generate loss-of-function mutations efficiently, sgRNA target sites were designed in the first half of the open reading frame, as close as possible to the start codons. According to those editing rules of Cas9-cytidine deaminase fusion in BsaI in pBmBE3. The oligonucleotides (each oligonucleotide was 70 bp) were synthesized by array-based oligonucleotide pool synthesis. Then all oligonucleotides were amplified by PCR. The PCR products were purified and ligated into the BsaI-digested pBmBE3 vector by Gibson ligation, and the successfully ligated plasmids were transformed into Psa or E. coli DH5α chemically competent cells  or CGA (Arg) codons were located at 20 bases downstream of the TTTN structure, this region could be designed as a target site.Psa strain was taken from an ultra-cold storage freezer (−80 °C) and cultured on King’s B medium (containing 50 μg mL−1 Rifampicin) at 25 °C for 48 h before usage. A single colony was inoculated in 5 mL of liquid KB and shaken at 200 rpm min−1 overnight at 25 °C. The bacterial culture (2 mL) was transferred to 50 mL of liquid KB and incubated in the same conditions until the OD600 reached 0.5. Cells were collected by centrifuging at 4 °C for 10 min at 10,000× g and resuspended with a 5 mL cold CaCl2 solution (20 mM). The supernatant was discarded, and the bacterial cells were resuspended with the buffer (0.7 mL of 20 mM CaCl2 and 0.3 mL of 50% glycerol) and then dispensed into each centrifuge tube with 100 μL. The competent cells were immediately frozen in liquid nitrogen for 5 min and stored at −80 °C (Supplementary Notes 2 and Psa cells were added to 0.5 mL of liquid KB (0.6 mM IPTG) and cultured at 25 °C for 2 h at 180 rpm. Cells were inculcated on the KB solid medium (containing 50 μg/mL gentamicin and 0.6 mM IPTG) for 2 days at 28 °C. A single colony was selected, and the target gene was amplified and sequenced to identify the mutation events.The Psa infection assays were based on previous procedures [A. chinensis var. chinensis ‘Hongyang’ tissue culture plantlets were cut and grown on Murashige and Skoog rooting medium without antibiotics in plates. Plantlets were grown in a plant growth chamber with a temperature of 24 ± 1 °C and a photoperiod of 16 h light/8 h dark. Kiwifruit plantlets (4 weeks old) were infected using a plant flooding method [Psa (containing empty vector) and mutants were grown in liquid LB (containing 50 μg/mL gentamicin) followed by overnight shaking and washing, and the cell density was adjusted to 1 × 107 CFU/mL. Silwet L-77  as a surfactant was added to the Psa suspension at 0.0025% (vol/vol). Kiwifruit plantlets were fully submerged in Psa suspension for 5 min and grown in a plant growth chamber with a temperature of 16 °C with 16 h/light and 10 °C with an 8 h/dark cycle. Leaves were collected at 20 days postinoculation (dpi). Leaf samples were washed with ddH2O three times and ground with 1 mL of sterile 10 mM MgSO4. The leaf homogenate was stored at 4 °C overnight, and the supernatant was diluted 10-fold and plated as 10 μL droplets on the LB medium with 50 μg/mL Gm. After 2 days of incubation at 25 °C, the CFU per cm2 leaf was ascertained from dilutions.ocedures . The axig method . Psa (coPsa pathogenic genes focused on the effector proteins of the T3SS in the sequenced strain PSA.AH.01. We referred to relevant literature regarding DC3000 and selected 10 effectors, namely, HopAO2, HopR1, HopAI1, AvrE1, HopM1, HopI1, AvrB4, HopQ1, HopZ3, and HopF2, which have been reported in the literature [In this paper, the screening of terature ,47,48,49


Background: The locoregional recurrence of breast cancer has been reduced due to the multidisciplinary approach of breast surgery, systemic therapy and radiation. Early detection and better surgical techniques contribute to an improvement in breast cancer outcomes. Purpose of Review: The purpose of this review is to have an overview and summary of the current evidence behind the current approaches to the locoregional treatment of breast cancer and to discuss its future direction. Summary: With improved surgical techniques and the use of a more effective neoadjuvant systemic therapy, including checkpoint inhibitors and dual HER2-directed therapies that lead to a higher frequency of pathologic complete responses and advances in adjuvant radiation therapy, breast cancer patients are experiencing better locoregional control and reduced local and systemic recurrence. De-escalation in surgery has not only improved the quality of life in the majority of breast cancer patients, but also maintained the low risk of recurrence. There are ongoing clinical trials to optimize radiation therapy in breast cancer. More modern radiation technologies are evolving to improve the patient outcome and reduce radiation toxicities. Due to the multidisciplinary approach to locoregional disease in breast cancer, the local recurrence and long-term outcome of breast cancer have improved. The decline in ipsilateral breast cancer recurrence is due to multiple factors including early detection, better surgical techniques, an effective neoadjuvant and adjuvant systemic therapy and adjuvant radiation. The improvement in local recurrence and long-term outcome has shifted the treatment toward an improvement in quality of life and decreased morbidities, such as lymphedema, which is achievable with de-escalation. There are ongoing clinical trials to de-escalate local therapy while omitting surgery and/or radiation in ductal carcinoma in situ and early-stage hormone-positive breast cancer in older adults ,2. In thTo understand where we are with treatment, we must review the past. The surgical treatment of breast cancer has evolved drastically since the Halsted radical mastectomy was established as the operation for breast cancer for most of the 20th century. The radical mastectomy included the breast, pectoralis major muscle and a level 1–3 axillary lymphadenectomy. Patients and surgeons began to voice dissatisfaction for this procedure in the mid1960s , compariThe evolution of a radical mastectomy to less surgery was the first step toward improvements in the morbidity from lymphedema, pain, decreased range of motion and function. With the knowledge gained in the NSABP B-04 trial and womenThere has been much debate on the adequate margin needed at the time of surgical resection for breast cancer. This has ranged greatly in the literature from no ink on tumor to margins > 1 cm. There has also been debate between invasive and non-invasive breast cancer. In 2014, a consensus statement was issued after a systematic review and meta-analysis across all subspecialties including surgical oncologists, medical oncologists, radiation oncologists and breast pathologists. The consensus is that no ink on tumor minimizes the risk of invasive breast cancer recurrence and a 2 mm margin is required for pure ductal carcinoma in situ (DCIS) ,10. The Through the twentieth century, we moved further from the radical mastectomy to partial mastectomy for early-stage breast cancer. Women were able to have outpatient surgery with minimal morbidity, although the standard was to perform a level 1 and 2 ALND for patients with invasive breast cancer. Moving from an ALND to a sentinel lymph node (SLN) biopsy to surgically stage the axilla in clinically node negative patients was another major step in the de-escalation of breast cancer surgical treatment. SLN biopsy was shown repeatedly to be safe in patients who had a negative SLN ,12,13. BThe results of the ACOSOG Z0011 trial have been widely adopted for patients undergoing a partial mastectomy with a clinically negative axilla and are considered a standard of care in the NCCN guidelines version 4.2023 for invasive breast cancer. Patients have greatly benefited from this shift in treatment. In a retrospective chart review conducted at the Memorial Sloan Kettering Cancer Center, they looked at how many axillary lymph node dissections were avoided based on the Z0011 criteria. Out of 793 patients, 84% underwent SLND alone while the remainder underwent ALND based on ≥3 SLN-positive, extra capsular extension and/or surgeon choice . Upon fuThe question of the surgical treatment of the axilla in patients who are undergoing a mastectomy is still ongoing. Patients included in the Z0011 trial all underwent breast conservation with adjuvant breast irradiation. The tangential fields of radiation treated level 1 of the axilla and part of level 2. Due to this, there was concern that those patients undergoing a mastectomy who may not qualify for post mastectomy radiation therapy would have their axilla undertreated if any disease was noted in their pathology. The AMAROS trial  8] comp comp8] cThere has been a conundrum for patients who undergo a mastectomy and are found to have axillary metastasis through H&E in their SLN. A multidisciplinary conversation is necessary to discuss the patient’s clinical scenario and the necessity of completion ALND versus axillary radiation versus both ALND and radiation therapy. There remain clinical scenarios where an ALND remains the standard of care. These include patients who present with a clinically positive axilla and do not undergo neoadjuvant systemic therapy, those with a failed SLN mapping, those with sentinel or axillary lymph nodes that remain positive after neoadjuvant chemotherapy, those with an axillary recurrence following previous breast cancer treatment, those with matted lymph nodes or gross disease at the time of SLNB, those with occult breast cancer only presenting with axillary metastasis, SLN-positive patients who fall out of the Z0011 criteria and those with inflammatory breast cancer (ASBrS consensus statement). Lymphedema is a concern for all patients undergoing a lymphadenectomy. This known morbidity has been reported through all clinical trials including this treatment of the axilla. In the AMAROS trial, although there was no difference in survival, there was a significant difference in morbidity for the patient in terms of lymphedema with the axillary radiotherapy having 11% lymphedema noted at 5 years compared to 23% in the ALND group. Singhal et al. reported on the use of a prophylactic lymphovenous bypass at the time of ALND for breast cancer. This showed a decrease in lymphedema from at least 20% to 3.1% in the patient population treated . FurtherThe evolution of breast surgery has not occurred in a silo. The majority of the credit has to be given to the quickly changing and improving systemic therapy that we have available to our patients. From the trials discussed above one can see that more surgery does not equate to a better OS or DFS. Improved systemic therapy is what has improved survival in the treatment of breast cancer. As we better understand the biology of individual breast cancers and can personalize treatment, pathologic complete responses (pCR) have become more common especially with utilizing dual HER2-directed therapy in HER2-positive breast cancer patients and the use of pembrolizumab, an immune checkpoint inhibitor in triple-negative breast cancer patients in a neoadjuvant setting ,19. The This cohort of patients was excluded from trials such as AMAROS and Z0011 as there was concern over the false negative rate of sentinel lymph node biopsy after neoadjuvant chemotherapy. The ACOSOG Z1071 trial set to evaluate the false negative rate of sentinel lymph node biopsy after neoadjuvant chemotherapy ,24. ThisACOSOG 71071 and multiple other trials  have shown similar results. Patients who are converted to a clinically negative axilla with neoadjuvant systemic therapy can undergo a Targeted Lymph node Dissection (TLND) at the time of surgery. If there is any residual cancer noted in the lymph nodes that are removed, a completion ALND is recommended. Using systemic therapy to de-escalate axillary surgery will save many patients the morbidity of an ALND. The alliance A11202 study compares patients who undergo neoadjuvant chemotherapy and have a positive SLN. Patients are randomized to either ALND with chest wall and local lymph node radiation or to radiation alone regardless of surgery. Johnson et al. recently reported the results of a feasibility study by the Exceptional Responders Clinical Trials Group to eliminate surgery. Thirteen patients with triple-negative or HER2-positive breast cancer who received neoadjuvant systemic treatment underwent a vacuum-assisted core biopsy post systemic therapy. Those with pCR  did not have any surgery, but they received whole breast radiation therapy. The median follow-up of 44.3 months did not reveal any ipsilateral breast tumor recurrence. The expansion phase of this study is ongoing ,26.p = 0.279), but radiation therapy did reduce recurrence  [The de-escalation of surgery in the axilla has been very important in reducing the morbidity of surgery. SLN biopsy is performed in all patients who present with a clinically negative axilla. In 2013, Hughes et al. published their results of the CALGB 9343 trial that looked at lumpectomy plus tamoxifen with or without irradiation in women aged 70 or older with early-stage breast cancer . They fo= 0.002) . The omiAlthough surgical techniques have changed drastically, there continues to be some morbidity that is unavoidable after surgery. Omitting surgery all together in some settings would be beneficial to patients if oncologically safe. Multiple retrospective trials ,31,32,33More recently, multiple prospective studies have utilized an image-guided core needle biopsy to evaluate for pCR using ultrasound and mammography ,36,37. Tp < 0.0003). Both hormone-receptor-positive and negative DCIS patients were enrolled in this study. There was no benefit from adding tamoxifen for hormone receptor-negative patients. At a 10-year follow-up, patients maintained low LRR [Multiple studies have shown the benefit of systemic therapy in reducing the locoregional recurrence (LRR) of breast cancer. NSABP B-24 was one of the first studies that was conducted and that showed a reduction in the rate of LRR with tamoxifen in patients with DCIS. Patients with DCIS were randomized to receive tamoxifen in an adjuvant setting after surgical resection and radiation therapy. At a 7-year follow-up, patients who received tamoxifen showed a 39% relative risk reduction of ipsilateral and contralateral breast cancer; both stage 0 and invasive cancer were reduced from 16% in the placebo group to 10% in the tamoxifen group  that randomized early-stage, node-negative breast cancer patients to lumpectomy and whole breast irradiation with or without systemic therapy showed lower LRR in patients who received systemic therapy. Of the patients who did not receive any systemic therapy, 12.3% had a 12-year incidence of ipsilateral recurrence compared to patients assigned to tamoxifen alone, chemotherapy alone, and tamoxifen plus chemotherapy of which 6.7%, 6.4%, and 6.8% had ipsilateral recurrences, respectively ,46. The secondary analysis of the TAILORx randomized clinical trial that was reported by Sparano et al. showed that patients with a high Oncotype DX recurrence score of 26 to 100 who received adjuvant chemotherapy did extremely well and 91% had a 5-year freedom of breast cancer distant or locoregional recurrence .Adjuvant radiation has been an integral component of breast conservation for several decades. In 2011, the Early Breast Cancer Trialist’ Collaborative Group (EBCTCG) meta-analysis showed that for node-negative patients, radiation reduced the risk of local recurrence from 31.0% to 15.6% at 10-years and reduced the 15-year risk of breast cancer death from 20.5% to 17.2% . For nodRegional nodal irradiation (RNI) is the radiation of the draining lymph nodes of the breast, which often includes the axilla (levels I–III), supraclavicular lymph nodes (SCL) and internal mammary lymph nodes (IMN), after either a mastectomy or breast conserving surgery. In the late 1990s, multiple trials established RNI after a mastectomy as the standard of care for women with node-positive or high-risk breast cancer ,50,51. IThe treatment of the regional nodes in women with 1–3 positive lymph nodes had been a source of controversy for several years. In 2011, the EBCTCG sought to end the debate with a meta-analysis that showed that the radiation after BCS reduced any first recurrence from 58.2% to 38.3%, for an absolute 10-year gain of 19.9% . In 2014Neoadjuvant chemotherapy (NAC) is increasingly being used in the treatment of breast cancer. NAC can not only downstage tumors, making them more amenable to breast-conserving surgery (BCS), but also a tumor’s response to NAC can give insight into the tumor biology and prognosis ,55. It iTwo large, randomized trials are currently investigating the role of radiation therapy after NAC in patients with a clinically node-positive disease. The NSABP B-51/RTOG 1304 trial  is evaluFollowing the practice changing TAILORX trial, molecular assays are increasingly being used to guide and individualize breast cancer treatment . PatientTwo randomized trials have been initiated that aim to use genetic biomarkers to guide the adjuvant radiation. NRG-BR007 , also knConventional whole breast radiation therapy is delivered in 25 fractions over 5 weeks. However, there is a growing interest in the use of non-standard radiation fractionations, such as hypofractionated whole breast radiation (HBRT) and ultra-hypofractionated whole breast radiation (UHBRT) to make radiation treatment more convenient for patients. The FAST-FORWARD trial compared HBRT (1 week) to conventional whole breast radiation (3 weeks) in patients with early-stage breast cancer. The trial found that HBRT was non-inferior to conventional whole breast radiation in terms of local control and toxicity . The NRGRadiation oncology is also moving towards more individualized treatments that maximize the cancer outcomes and minimize the treatment-related toxicity. Modern radiation techniques such as intensity-modulated radiation therapy (IMRT), prone positioning and proton therapy have reduced the dose to normal tissues such as the heart and lung, thereby minimizing any potential radiation-related cardiac or pulmonary toxicities. Smaller target volumes seen in partial breast irradiation further minimize the dose to normal tissues for lower-risk patients. Through appropriately de-escalating RNI in those patients who do not derive benefit, by omitting radiation in select lower-risk patients, modifying radiation targets to account for smaller surgeries and utilizing modern radiation treatment planning, radiation oncology is adapting and evolving to improve breast cancer treatment for all patients. In the future, more modern radiation technologies, such as FLASH radiotherapy, may be able to spare normal tissue while maintaining the anti-tumor effectiveness. Due to an improvement in surgical techniques, a neoadjuvant systemic therapy that includes checkpoint inhibitors in triple-negative breast cancer patients and dual HER2-directed therapy in HER2-positive breast cancer patients leads to an improved pCR plus advances in radiation therapy which have changed the outcome of breast cancer patients. With the increased use of neoadjuvant systemic therapy, breast cancer patients are having less aggressive surgeries. The management of axilla has changed tremendously. We can downstage the axilla and reduce the number of axillary node dissections even if the patient had clinically positive lymph nodes before starting the neoadjuvant chemotherapy, which turned into negative axilla post neoadjuvant chemotherapy. We are escalating neoadjuvant therapy by adding a checkpoint inhibitor (pembrolizumab) and dual HER2-directed therapy to neoadjuvant chemotherapy in triple-negative and HER2-positive breast cancer patients even though some studies have shown a de-escalation from the chemo portion of neoadjuvant systemic therapy in selected HER2-positive patients can maintain the high pCR rate, which is translated into an improvement in DFS and OS and a decrease in treatment-associated toxicities . Breast cancer patients are experiencing less locoregional and systemic recurrences and an improvement in DFS and OS. Early-stage breast cancer patients with proper surgery, adjuvant systemic therapy and radiation have a 0.5% risk of local recurrence per year, which previously was reported as 1% . The useThere will always be a role for breast surgery in the treatment of breast cancer, but we have shown repeatedly that less surgery is equivalent in regard to disease free and disease specific overall survival. The de-escalation of surgery has been made possible with the improvement of systemic therapy in a neoadjuvant setting. Adjuvant systemic therapy also plays an important role in reducing both the systemic and local recurrence of breast cancer. There are a number of controversies surrounding the use of radiation therapy in breast cancer. These controversies are being investigated in a number of clinical trials. Multiple ongoing studies are investigating how to utilize and validate biomarkers in the adjuvant radiation therapy of breast cancer patients. The results of these trials will help to inform the optimal use of radiation therapy in breast cancer. More modern radiation technologies are evolving to improve the patient outcome and reduce the radiation toxicities.The evolution for the loco-regional treatment of breast cancer has de-escalated in a short amount of time as systemic therapy has improved. This allows us to give patients more options and personalize their care. A robotic-assisted nipple sparing mastectomy and sensation sparing mastectomy are two new additions to our surgical arsenal to improve patients’ quality of life. In addition to omitting surgery all together, further studies are continuing to look at percutaneous treatment including cryoablation and radiofrequency ablation. We predict that the de-escalation/omission of radiation therapy in low-risk breast cancer patients will be on the horizon.


Introduction: Inosine monophosphate dehydrogenase 1 (IMPDH1) is a critical enzyme in the retina, essential for the correct functioning of photoreceptor cells. Mutations in IMPDH1 have been linked to autosomal dominant retinitis pigmentosa subtype 10 (adRP-10), a genetic eye disorder. Some of these mutations such as the Asp226Asn (D226N) lead to the assembly of large filamentous structures termed cytoophidia. D226N also gives IMPDH1 resistance to feedback inhibition by GDP/GTP. This study aims to emulate the adRP-10 condition with a long-term expression of IMPDH1-D226N in vitro and explore cytoophidium assembly and cell survival. We also assessed whether the introduction of an additional mutation (Y12C) to disrupt the cytoophidium has an attenuating effect on the toxicity caused by the D226N mutation.Results: Expression of IMPDH1-D226N in HEp-2 cells resulted in cytoophidium assembly in ∼70% of the cells, but the presence of the Y12C mutation disrupted the filaments. Long-term cell survival was significantly affected by the presence of the D226N mutation, with a decrease of ∼40% in the cells expressing IMPDH1-D226N when compared to IMPDH1-WT; however, survival was significantly recovered in IMPDH1-Y12C/D226N, with only a ∼10% decrease when compared to IMPDH1-WT. On the other hand, the IMPDH1 expression level in the D226N-positive cells was <30% of that of the IMPDH1-WT-positive cells and only slightly higher in the Y12C/D226N, suggesting that although cell survival in Y12C/D226N was recovered, higher expression levels of the mutated IMPDH1 were not tolerated by the cells in the long term.Conclusion: The IMPDH1-D226N effect on photoreceptor cell survival may be the result of a sum of problems: nucleotide unbalance plus a toxic long-life cytoophidium, supported by the observation that by introducing Y12C in IMPDH1 the cytoophidium was disrupted and cell survival significantly recovered, but not the sensibility to GDP/GTP regulation since higher expression levels of IMPDH1-D226N were not tolerated. Inosine monophosphate dehydrogenase 1 (IMPDH1) is a crucial enzyme in the retina that plays a critical role in the survival and function of photoreceptor cells, as the demand of cGMP for the phototransduction process is very high. These cells are responsible for converting light into electrical signals, and any dysfunction or loss of these cells can lead to severe vision impairments. In photoreceptor cells, the isoform 1 of IMPDH is predominant , probablin vivo to adjust GTP synthesis to different lighting conditions , a genetic eye disorder that affects approximately 1 in 4000 people . The adRin vivo adRP-10 patient condition with a long-term expression of IMPDH1-D226N in vitro and explore cytoophidium assembly and cell survival. We will also evaluate if by disrupting the cytoophidium with another mutation in residue Tyr12 , results in “permanent” cytoophidium assembly that cannot be disrupted by guanosine supplementation , meaningue Tyr12 , or by tue Tyr12 , toxicitß isoform (NCBI Reference Sequence: NM_000883.4) was PCR-amplified from a human cell line cDNA (HEp-2), with the following primers: F: atggcggactacctgatcagc; R: gtacagccgcttttcgtaagagtg, and the GMPR (NCBI Reference Sequence: NM_006877.4), with the following primers: F: atgccccgcatagatgcggacc; R: ttagctgaacacggtgttgtg. The genes were inserted into the linearized pCMV3 vector  that contains piggyBac inverted repeats for the transposon system, by using ClonExpress UItra One Step Cloning Kit  according to the manufacturer’s protocol. To the IMPDH1 gene, a myc tag was fused at the C′ terminus and a dark red fluorescent molecule  at the N′ terminus, interleaved by a P2A sequence. To the GMPR gene, a blue fluorescent protein BFP  was added at the N′ terminus, interleaved by a P2A sequence. The final IMPDH1 construct was pCMV_iRFP670_P2A_IMPDH1-Myc. The final GMPR construct was pCMV_BFP_P2A_GMPR.The IMPDH1 coding sequence for the canonical 514aa Site-directed mutagenesis was carried out by linearizing target constructs with primers containing individual point mutations, followed by recirculation of the plasmid with the Gibson assembly system and NEBuilder HiFi Master Mix Kit . Gene size, sequence, and point mutations were confirmed by agarose gel and Sanger sequencing.ß were replaced by the 38aa fragment  were grown to confluence with the culture medium DMEM supplemented with 2 mM L-glutamine and 1% antibiotic–antimycotic (Thermo Fisher Scientific), plus 10% fetal bovine serum . The cells were cultured in a 37°C humid incubator with 5% COAll transfections were carried out with TurboFect or Lipofectamine 3000 transfection reagents  diluted in Opti-MEM I Reduced Serum Medium , according to instructions provided by the manufacturer.For transient overexpression, plasmids were transfected alone or mixed (as indicated in the Results) and the cells analyzed 24–48 h after transfection. For generation of cell lines with stable overexpression of the proteins, plasmids were transfected together with a transposase vector , and after 48 h, the cells were selected with 100 μg/mL of hygromycin B for 2–3 weeks  before aFor preparation of the IMPDH inhibitor drugs, ribavirin 250 mg pills  were diluted in water and filtered (0.22 µm) for a 100 mM stock solution. For MPA, mycophenolate sodium 360 mg pills  were diluted in PBS and filtered to obtain a 100 mM stock solution. Drugs were added to the cells 4 h before fixation in a 1/1000 final dilution, or otherwise as indicated in the Results.For analysis of cytoophidium, the cells grown in 13-mm round coverslips were fixed with 4% paraformaldehyde and probed with antibodies in an indirect immunofluorescence assay, as previously described .Primary antibodies used: rabbit polyclonal anti-IMPDH2 antibody , mouse anti-Myc monoclonal antibody 9E10 , and mouse anti-Flag monoclonal antibody clone M2 . Secondary antibodies used: Cy3-conjugated donkey anti-mouse IgG  and Alexa Fluor 488-conjugated donkey anti-rabbit IgG . After immunofluorescence labeling, the cells were covered with VECTASHIELD containing DAPI , and the images were captured either by a fluorescence microscope with 200 × or 400 × magnification  or by a confocal microscope .Cytometry analysis of the reporter genes (iRFP670 and BFP) was performed as previously described . BrieflyTwo methods were applied for analyzing apoptosis. First, the cells were labeled with Annexin V conjugated to Alexa Fluor 488 and propidium iodide , following the manufacturer`s protocol.Apoptosis was also measured by transfecting the cells 24 h before analysis with a plasmid containing a caspase-3 cleavage sequence, named FlipGFP, as detailed elsewhere . After cAs a positive control, the cells were treated with 0.5 µM of staurosporine for 4 h before analysis. In addition, another positive control was induction of apoptosis by exposing the bare cells to UV light for 1 min, followed by analysis 24 h later.Images captured using the microscope were analyzed using ImageJ 1.53 software. The proportion of cells presenting a given characteristic, such as cytoophidium, was obtained by quantification of at least two randomly captured images (>100 cells) in each of at least two independent experiments.All cytometry data were analyzed with CytExpert v2.3 software. Plasmid construction was designed, and DNA sequencing results were analyzed with SnapGene v3.2.1 software.t-test with Welch`s correction was applied. If the data require sample pairing, first the repeated measures one-way ANOVA (RM-ANOVA) was applied to assess if the difference among the groups was significant. If it was, comparisons among two groups were made by a two-tailed paired t-test  or Wilcoxon matched-pairs signed rank test . Normal distribution was evaluated by the D'Agostino and Pearson normality test.The data are presented as mean plus error bars indicating standard deviation (S.D.) or standard error of the mean (S.E.M.) as described in the figure legends. For statistical comparisons, if the data did not require sample pairing, an unpaired two-tailed p ≤ 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism v7.0 software.Comparisons among each condition and the reference (IMPDH1-WT) are shown above each data-bar, as indicated by the red arrows in the graphs . Other cTo better understand the cytoophidium assembly behavior of IMPDH1-D226N, as well as the potential of replacing Tyr12 for another residue , in thisOverexpression of IMPDH1-D226N mutation induces cytoophidium in ∼70% of transfected cells, significantly higher than that of IMPDH1-wt overexpression. Curiously, under ribavirin or MPA treatment, the cytoophidium turns into large clumps in ∼50% of the cells , as shown in Since the reporter gene was fused to the IMPDH1 interleaved by a P2A sequence, by analyzing the iRFP670 mean fluorescence intensity (MFI) in the positive cells, we could estimate the expression level of IMPDH1, estimating the amount of protein that was not toxic enough to prevent long-term cell survival. The average MFI in the D226N-positive cells was <30% of that of the IMPDH1-WT-positive cells . AltogetWe then evaluated if the presence of Y12C mutation that disrupts cytoophidium  could deHowever, when we analyzed the MFI and the percent of high iRFP670, there was only a slight although significant increase in the Y12C/D226N compared with the D226N alone , with vaWe hypothesized that the improved cell survival in Y12C/D226N when compared with the D226N alone could be, in part, credited to the presence of the cytoophidium-disrupting Y12C since in all analyses of percent of iRFP670 and MFI, the cells expressing IMPDH1-Y12C tolerate slightly increased expression levels of protein when compared with IMPDH1-WT .One of the hypotheses for the toxic effect of D226N mutation on IMPDH1 is that it may disrupt feedback inhibition of IMPDH1 by GDP/GTP , which iIn the long-term cell-lines, percent of D226N-expressing cells among the GMPR-WT positive cells was 42.5% ; this reSimilar to when expressing mutated IMPDH1 alone, in the presence of GMPR, percent of high iRFP670 presented a slight although significant increase for Y12C/D226N, but still significantly lower than that of IMPDH1-WT + GMPR-WT . In addiEither an accumulation of protein aggregates or an unbalanced pool of nucleotides would result in cell damage and death by apoptosis. To evaluate the dynamics of cell death induced by the presence of D226N without the effect of hygromycin B treatment, the percent of iRFP670-expressing cells was analyzed 24 h after transfection (T1) and within 3-day intervals (time-points) up to 2 weeks . Within To measure apoptosis, we used a plasmid containing a caspase-3 cleavage sequence, named FlipGFP, as detailed elsewhere . The gatIn the cells with stable expression of IMPDH1-WT or D226N, apoptosis was labeled for phosphatidylserine translocation with Annexin V . In all Altogether, the presence of IMPDH1-D226N results in a ∼20–30% increase in the apoptosis rate, with a cumulative effect, meaning within 1 week, the percent of iRFP670-positive cells will reach the ∼40–50% decrease observed in the D226N cell lines when compared with IMPDH1-WT.ß isoform, with 514aa. However, in human retinal photoreceptor cells, an alternative splicing isoform α is the predominant IMPDH1 isoform expressed   without hygromycin selection . A total (day 7) . In addionical ß .Altogether, these data show that cell survival was slightly lower in the presence of IMPDH1-α(546aa) when compared with the canonical IMPDH1-β(514aa), a ∼10% decrease, but the intensity of damage is much less severe than that of IMPDH1-D226N. However, since α(546aa) is the major isoform in the human retina, in adRP-10 patients that carry the IMPDH1-D226N mutation, the effect would add up and damage photoreceptor cells even further.in vivo side effect of this mutation in retinal photoreceptor cells. We were also able to significantly recover cell survival by introducing another mutation in IMPDH1, the Y12C, which disrupts the filaments.Our data show that the adRP-10-related IMPDH1 missense mutation D226N induces formation of large cytoophidium, and the replacement of tyrosine at position 12 disrupts those cytoophidia, as previously demonstrated elsewhere . The lonThe impact of the exogenous IMPDH1-Y12C on the polymerization of endogenous IMPDH2 and the assembly of the cytoophidium, which is known as a large bundle of IMPDH polymers, can be attributed to the fundamental understanding that IMPDH polymers are composed of octamers . Given tin vivo model that faithfully replicates adRP-10-related IMPDH1 mutations poses significant challenges due to the difficulty of introducing mutations in animals and waiting for the disease phenotype to manifest. In vitro, the complex metabolism within photoreceptor cells is also a challenge, as well as retinal cell lines such as the retinal pigment epithelial cell (RPE1) or the 661W cone photoreceptor cell line which grow at a much slower rate than other more traditional tumor-derived cells, such as HeLa and HEK293-T. In our model, we used HEp-2 cells, initially thought to be a lineage of laryngeal carcinoma, but recent evidence suggests that it is derived from a HeLa contamination (α is the predominant IMPDH1 isoform, with 546aa. IMPDH1-α has decreased sensitivity to GTP feedback inhibition when compared with the canonical 514aa IMPDH1-β (Creating an accurate mination . In any mination . HoweverIMPDH1-β . Our resBy utilizing this model, we could emulate the long-term expression of IMPDH1-D226N and evaluate cytoophidium assembly, cell tolerance, and survival. However, there are at least 12 mutations in IMPDH1 that are associated to adRP in humans . From thIn retinal cells, the presence of cytoophidium is tightly regulated by light conditions, as demonstrated in previous studies . This rein vivo. The D226N-induced “toxic long-life cytoophidium” hypothesis would be in addition to losing its sensibility to GDP/GTP inhibition (in vivo using an adRP-10 animal model.In conclusion, we hypothesize that since cytoophidium increases protein half-life by making it difficult to be degraded , this cohibition , which rhibition . Indeed,hibition , as well