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Background: Breast conserving surgery (BCS) is typically followed by adjuvant radiotherapy (RT) based on several landmark trials demonstrating improvements in disease control and survival. Since completion of these historical trials, the advent of molecular subtyping has revealed that breast cancer is not a single disease entity, but rather a class of cancers with differential risk profiles. We evaluated whether RT could be safely omitted following BCS for patients with the most favorable subtype as defined by the Prosigna PAM50 assay. Methods: We conducted a multicenter prospective single-arm cohort study with IRB approval and an FDA investigational device exemption (IDE). Eligible patients were women 50 to 75 years of age (inclusive) who had undergone BCS revealing tumors ≤2cm in size, that were estrogen or progesterone receptor positive (HR+), HER2 negative, grade 1-2, node negative (N0), with negative excision margins (no ink on tumor). Intent to take endocrine therapy was required. Upon registration, tumors were submitted for central Prosigna testing and those with Risk of Recurrence (ROR) score ≤40 were deemed eligible for the investigational omission of RT. The primary endpoint was the 5-year locoregional recurrence rate (LRR). Anticipating a total of 345 RT-omitting patients to enroll over 3.5 years, the study was designed with 90% power to exclude a 5-year LRR of 5% using a one-sample exponential test with one-sided type I error of 0.025. Results: From 2016 to 2020, 671 patients were registered from 13 centers, inclusive of affiliated regional network sites. Of these, 382 patients had a ROR Score ≤40 and opted to forego RT, comprising the main intention-to-treat (ITT) study population. Median age was 65 years (range 50 to 75), and median tumor size was 0.9 cm (range 0.1 to 2.0 cm). At a median follow-up of 26.9 months, 12 events were observed: 4 patients had ipsilateral in-breast recurrences, 7 had contralateral breast cancers, and 1 developed an unrelated melanoma. There were no regional-nodal or distant recurrences. The 2-year cumulative rate of LRR was 0.3% (95% CI: 0 – 1.0%). Of the 4 ipsilateral breast recurrences, 2 were in the same quadrant as the original primary tumor. Conclusion: In this preliminary report of the PRECISION trial, patients 50-75 years of age undergoing BCS and endocrine therapy for pT1N0 HR+ HER2-negative breast cancer with ROR score ≤40 had exceedingly low rates of LRR in the absence of adjuvant RT at a median follow-up of 26.9 months. Additional follow-up is required to determine whether these favorable results are durable. Citation Format: Lior Z. Braunstein, Julia Wong, Deborah A. Dillon, Yu-Hui Chen, Paul Catalano, Oren Cahlon, Mahmoud B. El-Tamer, Rachel Jimenez, Atif Khan, Carmen Perez, Rinaa Punglia, Ron Shiloh, Laura Warren, David Wazer, Jean Wright, Elizabeth Buckley, Tari King, Simon Powell, Eric Winer, Jennifer Bellon. Preliminary report of the PRECISION Trial (Profiling Early Breast Cancer for Radiotherapy Omission): A Phase II Study of Breast-Conserving Surgery Without Adjuvant Radiotherapy for Favorable-Risk Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-12-02.

Introduction: Since the discovery of the cholinergic anti-inflammatory pathway, vagus nerve stimulation (VNS) has been suggested as a treatment option for chronic inflammatory conditions. This pathway relies on efferent vagal nerve fibers. Invasive cervical VNS activates afferent and efferent vagal nerve fibers and, thus, has the potential to directly activate the cholinergic anti-inflammatory pathway. In contrast, transcutaneous auricular VNS (taVNS) activates the auricular branch of the vagus nerve that is purely afferent and projects to the nucleus of the solitary tract. Thus, any anti-inflammatory actions of taVNS are unlikely to result from a direct activation of the cholinergic anti-inflammatory pathway. The objective of this study was to investigate alternative mechanisms by which taVNS may elicit anti-inflammatory effects. The hypothesis was tested that taVNS activates the hypothalamic-pituitary-adrenal (HPA) axis, resulting in adrenal cortisol secretion that will then modulate circulatory immune cell numbers. Methods: The study was approved by the appropriate authorities (IRB# 0054_2019 and NCT04177264). Study participants were healthy adults (n=19, 6 male, 13 female). On three consecutive days either taVNS (left ear, 10 Hz, 1-2 mA, 300 μs pulse width) or sham-taVNS (stimulator off) was applied for 10 minutes. A blood sample was drawn at the end of the third day. Plasma cortisol was determined by ELISA (K003-H1W, Arbor Assays, MI). Circulating T-helper cells (CD3+, CD4+), cytotoxic T-cells (CD3+, CD8+), B-cells (CD19+), monocytes (CD14+, CD11b+), and natural killer (NK, CD3-, CD56+) cells were counted using flow cytometry. Statistical significance was assumed at P Funding: This study was supported by a grant from the American Osteopathic Association (Grant No.: 19137759). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Postmastectomy radiation therapy (PMRT) is a mainstay of local therapy for many breast cancer patients. Patients undergoing mastectomy typically are offered options for breast reconstruction. For patients who are candidates for PMRT, there are ongoing challenges with combining optimal radiation technique to prioritize oncologic outcomes, against the goals of minimizing toxicity and achieving the best reconstruction outcomes. The process by which these decisions are made continues to evolve as surgical and radiation techniques have improved and expanded, and as more patients with different risk profiles for local recurrence are receiving PMRT. We review the considerations regarding the different types of reconstruction and timing with radiation treatment. We also review the technical radiation consideration such as dose-fractionation, use of bolus and scar boost, exploring the controversies associated with the nuanced decisions regarding radiation and reconstructive surgery which are influenced by variables that are under continued investigation.

sgRNA template assembly, in vitro T7transcription, and sgRNA column cleanup to remove 5'-triphosphate groups

Alveolar formation increases the surface area for gas exchange. A molecular understanding of alveologenesis remains incomplete. Here, we show that the autonomic nerve and alveolar myofibroblast form a functional unit in mice. Myofibroblasts secrete neurotrophins to promote neurite extension/survival, whereas neurotransmitters released from autonomic terminals are necessary for myofibroblast proliferation and migration, a key step in alveologenesis. This establishes a functional link between autonomic innervation and alveolar formation. We also discover that planar cell polarity (PCP) signaling employs a Wnt-Fz/Ror-Vangl cascade to regulate the cytoskeleton and neurotransmitter trafficking/release from the terminals of autonomic nerves. This represents a new aspect of PCP signaling in conferring cellular properties. Together, these studies offer molecular insight into how autonomic activity controls alveolar formation. Our work also illustrates the fundamental principle of how two tissues (e.g., nerves and lungs) interact to build alveoli at the organismal level.

Alveolar formation requires coordinated movement and interaction between alveolar epithelial cells, mesenchymal myofibroblasts, and endothelial cells/pericytes to produce secondary septa. These processes rely on the acquisition of distinct cellular properties to enable ligand secretion for cell-cell signaling and initiate morphogenesis through cellular contraction, cell migration, and cell shape change. In this study, we showed that mitochondrial activity and distribution play a key role in bestowing cellular functions on both alveolar epithelial cells and mesenchymal myofibroblasts for generating secondary septa to form alveoli in mice. These results suggest that mitochondrial function is tightly regulated to empower cellular machineries in a spatially specific manner. Indeed, such regulation via mitochondria is required for secretion of ligands, such as platelet-derived growth factor, from alveolar epithelial cells to influence myofibroblast proliferation and contraction/migration. Moreover, mitochondrial function enables myofibroblast contraction/migration during alveolar formation. Together, these findings yield novel mechanistic insights into how mitochondria regulate pivotal steps of alveologenesis. They highlight selective utilization of energy in cells and diverse energy demands in different cellular processes during development. Our work serves as a paradigm for studying how mitochondria control tissue patterning.The lungs display an intricate, tree-shaped structure which enables the complex gas exchanges required for life. The end of each tiny ‘branch’ hosts delicate air sacs, or alveoli, which are further divided by internal walls called septa. In mammals, this final structure is acquired during the last stage of lung development. Then, many different types of cells in the immature alveoli multiply and reach the right location to start constructing additional septa. While the structural changes underlining alveoli maturation are well-studied, the energy requirements for that process remain poorly understood. In particular, the exact role of the mitochondria, the cellular compartments that power most life processes, is still unclear. Zhang et al. therefore set out to map, in detail, the role of mitochondria in alveolar development. Microscope imaging revealed how mitochondria were unevenly distributed within the lung cells of newborn mice. Mitochondria accumulated around the machinery that controls protein secretion in the epithelial cells that line the air sacs, and around the contractile apparatus in the underlying cells (the ‘myofibroblasts’). Genetically altering the mice to reduce mitochondrial activity or perturb mitochondrial location in these two cell types produced defective alveoli with fewer septa, but it had no effect on lung development before alveoli formation. This suggests that the formation of alveoli requires more energy than other steps of lung development. Disrupting mitochondrial activity or location also compromised how epithelial cells produced chemical signals necessary for the contraction or migration of the myofibroblasts. Together, these results highlight the importance of tightly regulating mitochondrial activity and location during lung patterning. In the future, this insight could lay the groundwork to determine how energy requirements in various tissues shape other biological processes in health and disease.

Tibia fracture (BF) enhances stroke injury and post-stroke memory dysfunction in mouse. Reduction of neuroinflammation by activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) reduced acute neuronal injury and sensorimotor dysfunction in mice with BF 1-day after stroke. We hypothesize that reduction of neuroinflammation by activation of α-7 nAchR improves long-term memory function of mice with BF 6-h before stroke. The mice were randomly assigned to saline, PHA-568487 (α-7 nAchR agonist) and methyllycaconitine (antagonist) treatment groups. The sensorimotor function was tested by adhesive removal and corner tests at 3 days, the memory function was tested by Y-maze test weekly for 8 weeks and novel objective recognition test at 8 weeks post-injuries. We found PHA-568487 treatment reduced, methyllycaconitine increased the number of CD68+ cells in the peri-infarct and hippocampal regions, neuronal injury in the infarct region, sensorimotor and long-term memory dysfunctions. PHA-568487 treatment also reduced, while methyllycaconitine treatment increased atrophy of hippocampal granule cell layer and white matter damage in the striatum. In addition, PHA-568487 treatment increased neuron proliferation in granule cell layer. Our data indicated that reduction of neuroinflammation through activation of α-7 nAchR decreased neuronal damage, sensorimotor and long-term memory dysfunction of mice with BF shortly before stroke.

sgRNA template assembly, in vitro T7transcription, and sgRNA column cleanup to remove 5'-triphosphate groups

RationaleMutation in human arteriovenous malformation (AVM) causative genes in a fraction of endothelial cells (ECs) causes AVMs in mice. It is unclear how a small number of mutant ECs can lead to AVM formation.ObjectiveTo understand how a fraction of mutant ECs causes AVM, we tested the following hypotheses: (1) activin receptor-like kinase 1 (Alk1 or Acvlr1) mutant brain ECs undergo clonal expansion upon angiogenic stimulation, (2) Alk1 mutant ECs display growth advantage, (3) the burden of Alk1 mutant ECs correlates with AVM severity, and (4) Alk1 mutant bone marrow (BM) derived ECs alone is sufficient to cause AVM.Methods and ResultsWe used PdgfbiCreER;Alk1f/f;confetti+/− mice which express an EC-specific tamoxifen (TM)-inducible Cre recombinase, a Cre-regulated confetti transgene, and Alk1 floxed alleles. Brain AVMs were induced by direct brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) followed with intra-peritoneal injection of TM two weeks later. Color-predominance of confetti reporter in AVMs compared to control brain ECs suggested that clonal expansion was associated with AVM development. We treated PdgfbiCreER;Alk1f/f with different doses of TM to create a mosaic of wild-type (WT) and mutant ECs and found that equal numbers of Alk1+ and Alk1− ECs were proliferating. Increase of TM dose increased the number of Alk1− ECs, the abnormal vessels in brain AVMs, the number of arteriovenous shunts in the intestines, and mouse mortality. To test if mutation of Alk1 in BM-derived ECs can cause brain AVM, we transplanted WT mice with BM of PdgfbiCreER;Alk1f/f mice. After AAV-VEGF and TM treatment, these mice developed AVMs in their brains and arteriovenous shunts in their intestines.ConclusionClonal expansion of Alk1 mutant ECs could partly explain why a fraction of mutant ECs causes AVM. Mutation of AVM causal genes in BM-derived ECs is sufficient to cause AVM formation.

Alveolar formation increases the surface area for gas-exchange and is key to the physiological function of the lung. Alveolar epithelial cells, myofibroblasts and endothelial cells undergo coordinated morphogenesis to generate epithelial folds (secondary septa) to form alveoli. A mechanistic understanding of alveologenesis remains incomplete. We found that the planar cell polarity (PCP) pathway is required in alveolar epithelial cells and myofibroblasts for alveologenesis in mammals. Our studies uncovered a Wnt5a–Ror2–Vangl2 cascade that endows cellular properties and novel mechanisms of alveologenesis. This includes PDGF secretion from alveolar type I and type II cells, cell shape changes of type I cells and migration of myofibroblasts. All these cellular properties are conferred by changes in the cytoskeleton and represent a new facet of PCP function. These results extend our current model of PCP signaling from polarizing a field of epithelial cells to conferring new properties at subcellular levels to regulate collective cell behavior.

Introduction: Tibia fracture (BF) enhances stroke injury and when occurring 6 hrs before stroke (BF6+Stroke) causes long-lasting cognitive dysfunction in mouse. Activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) reduced neuroinflammation, neuronal injury and sensorimotor dysfunction in mice with BF one day after stroke (Stroke+1BF). Hypothesis: Activation of α-7 nAchR improves long-term cognitive function of BF6+Stroke mice. Methods: BF6+Stroke mice were randomly assigned to saline, PHA-568487 (α-7 nAchR agonist) and MLA (α-7 nAchR antagonist) treatment groups. The sensorimotor function were tested by adhesive removal and corner tests at 3 days, the cognitive function was tested by Y-maze weekly for 8 weeks and Novel Objective Recognition (NOR) at 8 weeks post-injuries. The neuronal damage, neuroinflammaiton, neurogenesis were analyzed 3 and/or 8 weeks post-injuries. Results: Similar to Stroke+1BF mice, PHA reduced and MLA enhanced neuronal injury, neuroinflammation, and sensorimotor dysfunction of BF6+Stroke mice. Further, PHA reduced and MLA enhanced their long-term cognitive dysfunction. In Y maze test, all mice made fewer alternations 1-week post-surgeries than baseline; PHA group recovered to baseline at week 5 post-surgeries; saline and MLA groups continuously made fewer alternations throughout the 8-weeks. In NOR test, PHA group spent more time, MLA group spent less time than saline group on novel objects. Injection of BrdU in the 2 nd week post-surgeries labeled more neurons in the contralateral than in the ipsilateral dentate gyrus in all groups; PHA group had the most, MLA group had the least BrdU + neurons. Injection BrdU in the 7th week post-surgeries did not labeled any neuron. Conclusion: Activation of α-7 nAchR decreased neuronal damage and neuroinflammation, increased neurogenesis at the dentate gyrus of BF6+Stroke mice; and improved their sensorimotor and long-term cognitive function.

Background and Purpose: Tibia fracture (BF) causes long-lasting memory dysfunction in stroke mice, which is associated with microglia accumulation in the hippocampus ipsilateral to the stroke injury. The underlying mechanism is unclear. Hypothesis: BF exacerbates blood brain barrier (BBB) breakdown and fibrin extravasation in the hippocampus enhancing white matter damage of stroke mice. Method: C57 mice (8-weeks) were randomly assigned to BF, stroke (pMCAO), BF+stroke (BF 6h before stroke) and sham groups. The integrity of BBB, fibrin deposition and CD68 + cells infiltration in the hippocampus were analyzed 3 days and the white matter injury in the basal ganglia was analyzed 8 weeks after the surgeries. Results: Compared to BF group, stroke and BF+stroke groups had lower level of claudin-5, fewer pericytes, more extravascular fibrin and CD68 + cells in the ipsilateral side of stroke 3 days after the injuries. BF+stroke group had the lowest level of claudin-5, fewest pericytes, highest extravascular fibrin and most CD68 + cells among the three groups. BF+stroke group also had a lower level of claudin-5 and fewer CD13 + pericytes in the contralateral side than the other two groups. Compared to sham group, the white matter bundle areas in the basal ganglia were reduced in stroke and BF+stroke groups in both contralateral and ipsilateral sides 8 weeks after the injuries. Stroke and BF+stroke groups also has smaller white matter bundle areas in the ipsilateral than contralateral side, and the white matter bundle areas in the contralateral side of BF+stroke group were also smaller than stroke group. Conclusion: BF shortly before stroke causes long-lasting memory dysfunction in mice through enhancing BBB breakdown and fibrin extravasation in the hippocampus, which exacerbates neuroinflammation and white matter damage.

The after mapping of the axilla: radiotherapy or surgery (AMAROS) trial concluded that for patients with cT1-2 N0 breast cancer and one or two positive sentinel lymph nodes (SLNs), axillary radiotherapy (AxRT) provides equivalent locoregional control and a lower incidence of lymphedema compared with axillary lymph node dissection (ALND). The study prospectively assessed how often ALND could be replaced by AxRT in a consecutive cohort of patients undergoing mastectomy for cT1-2 N0 breast cancer. In November 2015, our multidisciplinary group agreed to omit routine intraoperative SLN evaluation for cT1-2 N0 patients undergoing upfront mastectomy and potentially eligible for postmastectomy radiation therapy (PMRT), including those 60 years of age or younger and those older than 60 years with high-risk features. Patients with one or two positive SLNs on final pathology were reviewed to determine whether PMRT including the full axilla was an appropriate alternative to ALND. From November 2015 to December 2016, 154 patients met the study criteria, and 114 (74%) formed the final study cohort. Intraoperative SLN evaluation was omitted for 76 patients (67%). Of these patients, 20 (26%) had one or two positive SLNs, and 14 of these patients received PMRT + AxRT as an alternative to ALND. Three patients returned for ALND, and three patients were observed. On univariate analysis, tumor size, LVI, number of positive lymph nodes, and receipt of chemotherapy were associated with receipt of PMRT. For the majority of patients with one or two positive SLNs, ALND was avoided in favor of PMRT + AxRT. With appropriate multidisciplinary strategies, intraoperative evaluation of the SLN and immediate ALND can be avoided for patients meeting the AMAROS criteria and eligible for PMRT.

Isl1 is required for two processes during hindlimb development: initiation of the processes directing hindlimb development in the lateral plate mesoderm and configuring posterior hindlimb field in the nascent hindlimb buds. During these processes, Isl1 expression is restricted to the posterior mesenchyme of hindlimb buds. How this dynamic change in Isl1 expression is regulated remains unknown. We found that two evolutionarily conserved sequences, located 3' to the Isl1 gene, regulate LacZ transgene expression in the hindlimb-forming region in mouse embryos. Both sequences contain GATA binding motifs, and expression pattern analysis identified that Gata6 is expressed in the flank and the anterior portion of nascent hindlimb buds. Recent studies have shown that conditional inactivation of Gata6 in mice causes hindlimb-specific pre-axial polydactyly, indicating a role of Gata6 in anterior-posterior patterning of hindlimbs. We studied whether Gata6 restricts Isl1 in the nascent hindlimb bud through the cis-regulatory modules. In vitro experiments demonstrate that GATA6 binds to the conserved GATA motifs in the cis-regulatory modules. GATA6 repressed expression of a luciferase reporter that contains the cis-regulatory modules by synergizing with Zfpm2. Analyses of Gata6 mutant embryos showed that ISL1 levels are higher in the anterior of nascent hindlimb buds than in wild type. Moreover, we detected a greater number of Isl1-transcribing cells in the anterior of nascent hindlimb buds in Gata6 mutants. Our results support a model in which Gata6 contributes to repression of Isl1 expression in the anterior of nascent hindlimb buds.

Alveolar formation increases the surface area for gas-exchange and is key to the physiological function of the lung. Alveolar epithelial cells, myofibroblasts and endothelial cells undergo coordinated morphogenesis to generate epithelial folds (secondary septa) to form alveoli. A mechanistic understanding of alveologenesis remains incomplete. We found that the planar cell polarity (PCP) pathway is required in alveolar epithelial cells and myofibroblasts for alveologenesis in mammals. Our studies uncovered a Wnt5a–Ror2–Vangl2 cascade that endows cellular properties and novel mechanisms of alveologenesis. This includes PDGF secretion from alveolar type I and type II cells, cell shape changes of type I cells and migration of myofibroblasts. All these cellular properties are conferred by changes in the cytoskeleton and represent a new facet of PCP function. These results extend our current model of PCP signaling from polarizing a field of epithelial cells to conferring new properties at subcellular levels to regulate collective cell behavior., eLife digest The lungs enable the exchange of gases between inhaled air and the bloodstream. This exchange happens in structures called alveoli, which have a large surface area that aids in efficient gas exchange. Shortly after birth in mice, or during the last few months before birth in humans, alveoli develop folds called secondary septa that increase their surface area and improve the efficiency of gas exchange. Several types of cells work together to form secondary septa. Surface cells called epithelia and underlying “myofibroblast” cells and small blood vessels must both communicate and move together to build the septa. The processes that control the formation of septa have not been fully studied. In other cases, a cell signaling pathway known as the planar cell polarity (PCP) pathway has been shown to help coordinate cell movements. The PCP pathway works by changing the cytoskeleton of cells, which is the series of protein fibers that give cells their shape and structure and the ability to move. Zhang et al. have now studied septa in mouse lungs and revealed how three genes – Wnt5a, Ror2 and Vangl2 – in the PCP pathway control this process. This pathway oversees changes to the cytoskeleton in both epithelial cells and myofibroblasts, helping the cells to change shape and move together to form septa. Unusually, the PCP pathway has different effects in different cells, rather than affecting all cells similarly. This is partly due to so-called PDGF signals from the epithelial cells that help to guide the growth and movement of myofibroblasts. This process is helped by the epithelial cells changing their shape to accommodate myofibroblasts during septa formation. Further analysis also showed reduced PCP signaling in patients with chronic obstructive pulmonary disease, also known as COPD. This could be a factor in the extensive lung damage seen in these patients. These findings help to explain a key lung development process and may provide new insights to understand lung diseases such as COPD.

Objective. To evaluate the effectiveness of peer mentoring in helping first year, first-generation college students at Dominican University of California (DUC) adapt to university life and navigate the occupational challenges experienced during the first year of college. Method. Sixty-seven students voluntarily completed an online survey, First Year College Experience (FYCE) Survey: Adaptation to University Life. Quantitative research determined the influence of peer mentoring on the students’ adaptation and occupational performance in their transition to college. Effectiveness was determined by: 1) sense of belonging, 2) developed academic and social skills, 3) adaptive responses and strategies used, and 4) overall satisfaction with the college experience. Results. FGS experienced a greater sense of belonging compared to non-FGS (p = 0.012). Mentored students gained more skill over time academically than students who did not use peer mentoring (p = 0.003). There was no statistical difference between FGS and non-FGS in the use of adaptive strategies (p = 0.484). There was a statistical difference in use of adaptive strategies between students who were mentored and non-mentored (p=0.025). Mentored students self-reported having more problem solving strategies when confronted with a challenge compared to non-mentored students. Conclusion. The results suggest that peer mentoring is effective in helping students develop adaptive strategies, academic skills, and increasing overall college satisfaction. Implications of this study suggest that peer mentoring designed specifically for FGS in their first year of college may help FGS develop adaptive skills and flexibility in their problem-solving strategies that enhance their occupational performance as college students.

Background: Tibia fracture (BF) before stroke shortly causes long-term post-stroke memory dysfunction in mice. The mechanism is unclear. We hypothesize that BF enhances neuroinflammation and blood brain barrier (BBB) breakdown in the hippocampus and white matter (WM) damage. Methods: Mice were assigned to groups: BF, stroke, BF+stroke (BF 6 h before stroke) and sham. BBB integrity was analyzed 3 days after the surgeries and WM injury was analyzed 3 days and 8 weeks after the surgeries. Results: Stroke and BF+stroke groups had more activated microglia/macrophages and lower levels of claudin-5 in the ipsilateral hippocampi than the BF group. BF+stroke group had the highest number microglia/macrophages and the lowest level of claudin-5 among all groups and had fewer pericytes than BF group. Stroke and BF+stroke groups had smaller WM areas in the ipsilateral basal ganglia than the sham group 8 weeks after the injuries. The BF+stroke group also had smaller WM areas in the ipsilateral than sham and BF groups 3 days after the injuries and in the contralateral basal ganglia than stroke and BF groups 8 weeks after the injuries. Conclusions: BF exacerbates neuroinflammation and BBB leakage in the hippocampus and WM damage in basal ganglia, which could contribute to the long-lasting memory dysfunction in BF+stroke mice.

Background: Radiation therapy (RT) following breast conserving surgery (BCS) is known to improve locoregional and distant control in women with invasive breast cancer; however, RT does pose significant cost and potential risk, including concern for cardiopulmonary toxicity and secondary malignancy. Randomized trials show that the omission of RT among older women with otherwise favorable-risk breast cancer does not affect overall survival (OS) at 10 years, and modestly increases the risk of local recurrence (LR). Nevertheless, there is little prospective support for RT omission in younger women. Moreover, risk stratification in the randomized prospective studies to date relies on limited methods with potentially low fidelity at distinguishing luminal subtypes. Enhanced molecular profiling techniques could better discern tumor biology and, in turn, more precisely assess risk. PAM-50 is an expression profile of 50 genes used to classify breast cancer intrinsic subtype with consequent prognostic implications. The goal of this study is to identify low-risk breast cancer patients with luminal A subtype who may feasibly omit RT from their therapeutic regimen. Methods: This is a phase II prospective cohort study (NCT02653755) designed to assess the safety of treatment de-escalation (omission of RT) in women with favorable-risk invasive breast cancer following BCS. Eligibility criteria include female patients with invasive breast cancer, age 50-75, with tumors less than or equal to 2cm, negative surgical margins, ER+ or PR+ and HER2- disease, grade 1 or 2, no lymph node involvement, and no plans for chemotherapy or biologic therapy, who are eligible for and willing to receive endocrine therapy. Tumor samples from eligible patients will undergo PAM-50 transcriptional profiling using the Nanostring Prosigna assay. A low Risk of Recurrence (ROR) score, corresponding to the luminal A subtype, will permit patients to forego radiotherapy should they so choose. The primary endpoint is 5-year risk of local or regional recurrence of invasive or in situ breast carcinoma within the ipsilateral breast or regional lymph nodes. Study enrollment began in May 2016. Citation Format: Mira A. Patel, Deborah A. Dillon, Giulia Digiovanni, Yu-Hui Chen, Paul Catalano, Carmen Perez, David Wazer, Jean Wright, Rachel Jimenez, Eric Winer, Julia Wong, Jennifer Bellon, Lior Z. Braunstein. PRECISION (Profiling early breast cancer for radiotherapy omission): A phase II study of breast-conserving surgery without adjuvant radiotherapy for favorable-risk breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT271.

Background: Young women with breast cancer (BC) often receive aggressive local therapy and are more likely to undergo (bilateral) mastectomy and receive postmastectomy radiation (RT) than older women. Our previous work suggested better quality of life (QOL) outcomes among women undergoing breast conserving therapy (BCT) vs. mastectomy, even with reconstruction (recon), although limited data was available regarding recon. In the current analysis, we sought to understand the impact of recon, accounting for RT, on long-term satisfaction and QOL. Methods: Between 10/2016-11/2017, we administered the BREAST-Q, a validated patient-reported outcomes measure designed to evaluate satisfaction and QOL after breast cancer surgery and recon, to 743 women in the Young Women’s Breast Cancer Study, a multi-site prospective cohort that enrolled women diagnosed (dx’d) at age < 40 from 2006-2016. Demographic and treatment information was obtained by surveys and chart review. Recon was defined as autologous, implant or complex (combination of autologous and implant). Mean BREAST-Q scores for each domain (breast satisfaction, physical, psychosocial, and sexual) were compared by recon types and stratified by receipt of RT; higher BREAST-Q scores (range: 0-100) indicate better QOL. Linear regression was used to identify predictors of BREAST-Q scores. Results: 584 women with Stage 0-3 BC completed the Breast-Q at a median of 5.7 [range 2-10] years from dx), 357 (61%) had mastectomy and recon and were included in the analytic sample. Median age at dx was 36 years; 85% had stage 0, 1 or 2 disease; 41% received RT and 76% underwent bilateral mastectomy. 90.5% underwent immediate recon; of which 65.5% were tissue expander placement. Definitive recon included autologous tissue in 16%; implants in 81% and complex recon in 3%. Mean BREAST-Q breast satisfaction, psychosocial, and physical well-being scores differed based on recon type accounting for RT (Table 1). Significantly lower scores for breast satisfaction were seen in women having implant +RT. Scores were not impacted by bilateral procedure, timing of recon, use of tissue expander, extent of axillary surgery, cancer stage or other treatment factors. In multivariate analyses, receipt of RT, lymphedema and implant-based recon were associated with lower breast satisfaction scores. Physical well-being scores were lower for those with complex recon, for non-White women, for women who reported being financially uncomfortable, for those with lymphedema, and for those 25, and for those who reported being financially uncomfortable. Sexual well-being scores were lower for women with a BMI >25 and for those who reported being financially uncomfortable. Conclusion Type of recon and receipt of radiation among young survivors who undergo mastectomy and recon for early breast cancer appears to have a differential impact on satisfaction and QOL. Our finding of lower breast satisfaction among young women who received RT, particularly after implant recon, underscores the importance of counseling regarding surgical options, especially for women who may receive RT after mastectomy and who are candidates for BCT. Table 1. Unadjusted BREAST-Q scores stratified by RTBREAST-Q DomainAutologous -RT N=29Autologous +RT N=27Implant -RT N=174Implant +RT N=113Complex N=13Global p-valueSatisfaction with Breasts, mean (sd)67 (19)63 (18)64 (18)53 (18)58 (22) Citation Format: Laura Dominici, Jiani Hu, Hee Jeong Kim, Tari King, Kathryn Ruddy, Rulla Tamimi, Jeffrey Peppercorn, Lidia Schapira, Virginia Borges, Stephen Come, Ellen Warner, Julia Wong, Ann Partridge, Shoshana Rosenberg. BREAST-Q satisfaction and quality of life outcomes in young women undergoing mastectomy and reconstruction for breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD6-1.

Small regulatory nonprotein-coding RNAs (sRNAs) have emerged as ubiquitous and abundant regulators of gene expression in a diverse cross section of bacteria. They play key roles in most aspects of bacterial physiology, including central metabolism, nutrient acquisition, virulence, biofilm formation, and outer membrane composition. RNA sequencing technologies have accelerated the identification of bacterial regulatory RNAs and are now being employed to understand their functions. Many regulatory RNAs require protein partners for activity, or modulate the activity of interacting proteins. Understanding how and where proteins interact with the transcriptome is essential to elucidate the functions of the many sRNAs. Here, we describe the implementation in bacteria of a UV-crosslinking technique termed CRAC that allows stringent, transcriptome-wide recovery of bacterial RNA-protein interaction sites in vivo and at base-pair resolution. We have used CRAC to map protein-RNA interaction sites for the RNA chaperone Hfq and ribonuclease RNase E in pathogenic E. coli, and toxins from toxin-antitoxin systems in Mycobacterium smegmatis, demonstrating the broad applicability of this technique.

sgRNA template assembly, in vitro T7transcription, and sgRNA column cleanup to remove 5'-triphosphate groups

Small regulatory nonprotein-coding RNAs (sRNAs) have emerged as ubiquitous and abundant regulators of gene expression in a diverse cross section of bacteria. They play key roles in most aspects of bacterial physiology, including central metabolism, nutrient acquisition, virulence, biofilm formation, and outer membrane composition. RNA sequencing technologies have accelerated the identification of bacterial regulatory RNAs and are now being employed to understand their functions. Many regulatory RNAs require protein partners for activity, or modulate the activity of interacting proteins. Understanding how and where proteins interact with the transcriptome is essential to elucidate the functions of the many sRNAs. Here, we describe the implementation in bacteria of a UV-crosslinking technique termed CRAC that allows stringent, transcriptome-wide recovery of bacterial RNA-protein interaction sites in vivo and at base-pair resolution. We have used CRAC to map protein-RNA interaction sites for the RNA chaperone Hfq and ribonuclease RNase E in pathogenic E. coli, and toxins from toxin-antitoxin systems in Mycobacterium smegmatis, demonstrating the broad applicability of this technique.

The RNA landscape of all sequenced bacteria is littered with regulatory noncoding small RNAs (sRNA). Understanding the functions of these sRNAs has lagged behind their identification, as few high-throughput approaches existed to capture sRNA interactions in vivo. Recently, methodologies have been described that allow for profiling of the sRNA interaction network facilitating systems-level analysis sRNA regulation. This chapter discusses recent advances in our understanding of sRNA function, technical advances that allow us to capture sRNA interactions in vivo, and the computational tools that allow meaningful conclusions to be drawn from these data.

Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore- and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.

Importance The use of a radiotherapy (RT) boost to the tumor bed after whole-breast RT (WBRT) for ductal carcinoma in situ (DCIS) is largely extrapolated from invasive cancer data, but robust evidence specific to DCIS is lacking. Objective To compare ipsilateral breast tumor recurrence (IBTR) in women with DCIS treated with vs without the RT boost after breast-conserving surgery and WBRT. Design, Setting, and Participants This retrospective analysis pooled deidentified patient-level data from 10 academic institutions in the United States, Canada, and France from January 1, 1980, through December 31, 2010. All patients had newly diagnosed pure DCIS (no microinvasion), underwent breast-conserving surgery, and received WBRT with or without the boost with a minimum of 5 years of follow-up required for inclusion in the analysis. Given the limited events after WBRT, an a priori power analysis was conducted to estimate the DCIS sample size needed to detect the anticipated benefit of the boost. Data were uniformly recoded at the host institution and underwent primary and secondary reviews before analysis. Sample size calculations (ratio of patients who received the boost dose to those who did not, 2:1; α = .05; power = 80%) estimated that 2982 cases were needed to detect a difference of at least 3%. The final analysis included 4131 patients (2661 in the boost group and 1470 in the no-boost group) with a median follow-up of 9 years and media boost dose of 14 Gy. Data were collected from July 2011 through February 2014 and analyzed from March 2014 through August 2015. Interventions Radiotherapy boost vs no boost. Main Outcomes and Measures Ipsilateral breast tumor recurrence. Results The analysis included 4131 patients (median [SD] age, 56.1 [10.9] years; range, 24-88 years). Patients with positive margins, unknown estrogen receptor status, and comedo necrosis were more likely to have received an RT boost. For the entire cohort, the boost was significantly associated with lower IBTR (hazard ratio [HR], 0.73; 95% CI, 0.57-0.94; P = .01) and with IBTR-free survival (boost vs no-boost groups) of 97.1% (95% CI, 0.96-0.98) vs 96.3% (95% CI, 0.95-0.97) at 5 years, 94.1% (95% CI, 0.93-0.95) vs 92.5% (95% CI, 0.91-0.94) at 10 years, and 91.6% (95% CI, 0.90-0.93) vs 88.0% (95% CI, 0.85-0.91) at 15 years. On multivariable analysis accounting for confounding factors, the boost remained significantly associated with reduced IBTR (HR compared with no boost, 0.68; 95% CI, 0.50-0.91; P = .01) independent of age and tamoxifen citrate use. Conclusions and Relevance This patient-level analysis suggests that the RT boost confers a statistically significant benefit in decreasing IBTR across all DCIS age groups, similar to that seen in patients with invasive breast cancer. These findings suggest that a DCIS RT boost to the tumor bed could be considered to provide an added incremental benefit in decreasing IBTR after a shared discussion between the patient and her radiation oncologist.

This protocol, based on published work, demonstrates how to delivery Cas9 RNP-based gene editing reagents to cultured mamallian cells by electroporation with a Lonza 4d Nucleofector. Consider consulting some of the following papers: 1. RNP delivery paper upon which this work is based (Open Access): https://elifesciences.org/content/3/e04766 2. Paper by an IGI post-doc that details the rationale behind HDR donor design: https://www.ncbi.nlm.nih.gov/pubmed/26789497

sgRNA template assembly, in vitro T7transcription, andSPRI bead cleanup

sgRNA template assembly, in vitro T7transcription, andSPRI bead cleanup

Background: Hypophosphatemia (HP) is frequent following liver resection, and thought to represent use of phosphate during liver regeneration. We sought to evaluate the association of post-hepatectomy HP with liver insufficiency and recovery. Methods: Liver resections were retrospectively reviewed from 2009 to 2012 at a single institution. We explored the relationship between HP (defined as serum phosphate ≤0.65 mmol/L), occurrence of initial liver insufficiency (ILI) [bilirubin >50 μmol/L, international normalized ratio (INR) >1.7 within 72 hours of surgery] and in-hospital recovery of ILI. Secondary outcomes included 30-day post-operative major morbidity (Clavien grade 3 and 4 complications), mortality, and re-admission. Results: Among 402 patients, 223 (55.5%) experienced HP and 64 (15.9%) met our definition of ILI, of which 53 (82.8%) recovered. Length of stay, 30-day post-operative major morbidity, mortality, and re-admission were similar between patients with and without HP. Among patients with ILI, 44 (68.8%) experienced HP. Following ILI, patients with HP recovered more often than those with NP (90.9% vs. 65.0%; P=0.03). Conclusions: In patients who experience post-hepatectomy ILI, HP is associated with improved recovery, potentially indicating more efficient liver regeneration. Further studies should explore the usefulness of post-hepatectomy HP as an early prognostic factor of recovery from ILI.