Breast cancer patients with a negative axillary ultrasound may have clinically significant nodal metastasis.

INTRODUCTION: The non-invasive nature of the preoperative axillary ultrasound (AUS) fits the current trend of increasingly conservative axillary management. Recent publications suggest that early disease patients with clinically and radiologically negative axillae do not require sentinel lymph node biopsy (SLNB). This study aims to determine the true extent of axillary node disease in negative preoperative AUS patients. METHODS: A 10-year breast cancer registry was reviewed to identify women with pathologically confirmed T1-2 invasive breast cancer and a negative preoperative AUS. Patients who received neoadjuvant chemotherapy were excluded. Combined positive lymph node count of SLNB ± ALND was used to determine total nodal burden (TNB). Axillae were classified into low nodal burden (LNB) defined as 1-2 positive nodes and high nodal burden (HNB) defined as ≥ 3 positive nodes. RESULTS: 762 patients with negative AUS were included. There were 46.9% and 53.0% T1 and T2 tumours, respectively. 76.9% were node negative (0 LN +), 18.9% had LNB (1-2 LN +) and 4.2% had HNB (≥ 3LN +). Specifically, HNB disease was seen in 2% of T1 tumours and 6.2 % of T2 tumours with a negative AUS. In multivariate analysis, T2 strongly associated with ≥ 3 positive ALNs (OR 2.66 CI 1.09-6.51 p = 0.03) as did lymphovascular invasion (OR 3.56 CI 1.52-8.30 p = < 0.01). CONCLUSION: This study shows that AUS in its current form cannot exclude HNB axillary metastasis to the extent of eliminating the need for surgical staging of the axilla. This may impact axillary local-regional recurrence and disease-free survival. We caution that a negative AUS has a rate of 4.2% of HNB. Therefore, in cases of negative AUS with a T2 tumour, we advocate continued use of SLNB.

Clinically guided core biopsy and cutaneous punch biopsy in the evaluation of breast lesions: a necessary test or an obsolete skill?

OBJECTIVE: The vast majority of breast cancers are diagnosed via image-guided procedures yet despite significant advances, imaging does not identify all breast malignancies. Clinically suspicious breast lesions with normal breast imaging remain a cause for concern. The aim of this study is to determine the diagnostic value of clinical core and cutaneous punch biopsies in the diagnosis of breast malignancy in clinically suspicious lesions with normal breast imaging. METHODS: All patients with suspicious clinical breast findings and normal imaging who underwent a clinical core and/or cutaneous punch biopsy from 2012 to 2019 were reviewed retrospectively. Patients with subsequent breast malignant diagnosis were analysed. RESULTS: A total of 283 biopsies (166 clinical core, 117 cutaneous punch) performed over the 7-year period were included in the analysis. A total of 263/283 (93%) yielded a benign outcome. A total of 2/283 (0.7%) yielded B3 lesions (probably benign). These lesions were benign on final surgical excision. A total of 18/283 (6.3%) yielded a malignant histopathology. Sixteen out of 18 were cutaneous punch biopsies, and 2/18 were clinical core biopsies. A total of 14/18 patients presented with nipple changes, while 4/18 had a palpable area of concern. Histopathological analysis demonstrated Paget's disease of the nipple in 8/18, invasive carcinoma in 9/18 out of which two represented a recurrence of breast malignancy. Cutaneous squamous cell carcinoma was diagnosed in 1/18. CONCLUSION: Clinical core and cutaneous punch biopsies remain a valuable tool in the diagnosis of breast cancer particularly in the management of clinically suspicious radiographically occult malignancies.

AJEDI in Science: Leveraging Instructor Communities to Create Antiracist Curricula.

Gateway college science courses continue to exclude students from science, disproportionately discriminating against students of color. As the higher education system strives to reduce discrimination, we need a deliberate, iterative process to modify, supplement, or replace current modalities. By incorporating antiracist, just, equitable, diverse, and inclusive (AJEDI) principles throughout course design, instructors create learning environments that provide an antidote to historically oppressive systems. In this paper, we describe how a community of microbiology instructors who all teach Tiny Earth, a course-based undergraduate research experience, created and rapidly integrated antiracist content and pivoted to an online format in response to the social unrest and pandemic of 2020. The effort strengthened an existing teaching community of practice and produced collective change in classrooms across the nation. We provide a perspective on how instructor communities of practice can be leveraged to design and disseminate AJEDI curriculum.

Value of Long-term Follow-up in Surgically Excised Lesions of Uncertain Malignant Potential in the Breast - Is 5 Years Necessary?

INTRODUCTION: B3 lesions are a heterogeneous group of breast lesions of uncertain malignant potential which usually require excision. The aim was to assess the efficacy of 5 years routine radiological or clinical follow-up of patients who had "high-risk" B3 lesions surgically excised, by analyzing recurrence and subsequent development of invasive/in-situ cancer. PATIENTS AND METHODS: A 10-year retrospective review from 2010 to 2019 was performed of B3 lesions diagnosed on core needle biopsy, including patients who proceeded to surgical excision with a high-risk lesion on final histology. The database recorded 6 specific B3 lesion categories: 1. Atypical ductal hyperplasia (ADH), 2. Radial scars/complex sclerosing lesions (CSLs) with epithelial atypia 3. Classical Lobular neoplasia (ALH/LCIS), 4. Papillary lesions with epithelial atypia, 5. Mixed, 6. Flat epithelial atypia (FEA), including radiological and clinical follow-up data. RESULTS: Six hundred sixteen patients had a B3 lesion after core biopsy. 110 patients had "high risk" lesions. This included 17 (15.5%) Atypical Ductal Hyperplasia (ADH), 22 (20%) radial scars/CSLs with epithelial atypia, 47 (42.7%) classical lobular neoplasia (LCIS/ALH), 7 (6.4%) papillary lesions with epithelial atypia, 13 (11.8%) mixed lesions & 4 (3.6%) Flat Epithelial Atypia (FEA) lesions. 4 of 110 (3.6%) developed invasive/in-situ disease and 4 of 110 (3.6%) developed recurrence during follow-up. 33 of 616 (5.4%) upgraded to invasive/preinvasive disease after surgical excision. CONCLUSION: Five years of routine radiological surveillance may not be necessary in patients who undergo surgical excision of "high-risk" B3 lesions. Clinical surveillance appears to be of little benefit, especially in patients with radial scars, papillary lesions, and FEA. Subsequent development of invasive/in-situ disease in patients who undergo surgical excision of atypical B3 lesions remains low.

The OCT RNFL Probability Map and Artifacts Resembling Glaucomatous Damage.

Purpose: The purpose of this study was to improve the diagnostic ability of the optical coherence tomography (OCT) retinal nerve fiber layer (RNFL) probability (p-) map by understanding the frequency and pattern of artifacts seen on the p-maps of healthy control (HC) eyes resembling glaucomatous damage. Methods: RNFL p-maps were generated from wide-field OCT cube scans of 2 groups of HC eyes, 200 from a commercial normative group (HC-norm) and 54 from a prospective study group, as well as from 62 patient eyes, which included 32 with early glaucoma (EG). These 32 EG eyes had 24-2 mean deviation (MD) better than -6 dB and perimetric glaucoma as defined by 24-2 and 10-2 criteria. For the HC groups, "glaucoma-like" arcuates were defined as any red region near the temporal half of the disc. Results: Seven percent of the 200 HC-norm and 11% of the 54 HC RNFL p-maps satisfied the definition of "glaucoma-like," as did all the patients' p-maps. The HC p-maps showed two general patterns of abnormal regions, "arcuate" and "temporal quadrant," and these patterns resembled those seen on some of the RNFL p-maps of the EG eyes. A "vertical midline" rule, which required the abnormal region to cross the vertical midline through the fovea, had a specificity of >99%, and a sensitivity of 75% for EG and 93% for moderate to advanced eyes. Conclusions: Glaucoma-like artifacts on RNFL p-maps are relatively common and can masquerade as arcuate and/or widespread/temporal damage. Translational Relevance: A vertical midline rule had excellent specificity. However, other OCT information is necessary to obtain high sensitivity, especially in eyes with early glaucoma.

An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect.

Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(-)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.

Teaching "Crafty Microbiology": Safely Teaching Hands-On Microbiology Skills at Home.

Using nontoxic craft items and disposable lab consumables, we have developed nine modules to teach fundamental, hands-on microbiology lab skills safely at home. These "Crafty" teaching modules can be paired with virtual instruction and/or data collected by an instructor to replicate traditional microbiology lab exercises that characterize an unknown microbe. Materials and procedures used were carefully chosen to best mimic the texture of media, represent microbial diversity, assess aseptic technique, and produce analyzable data from results. Some protocols build upon and extend previously unpublished ideas, while others provide novel methods. The lab skills include proper personal protective equipment usage and basic biosafety, aseptic technique, microscopy and staining, streaking for isolation, spread plating, serial dilutions, filtering, disk diffusion method, and modeling an epidemic. Each protocol includes a student handout with background, links to videos of the methods performed with microbes, a rationale for the pairing of craft and consumable lab supplies along with technique used, a video or image demonstration of the "Crafty" technique when needed, postlab questions, and an instructor guide. This resource was developed for an undergraduate microbiology course, and each lab is aligned with learning outcomes within the American Society for Microbiology's undergraduate curriculum guidelines. This work would also be useful for outreach and K-12 educators. The development of microbiology lab skills by all students, regardless of economic or health status, will lead to a more scientifically minded society.

Phase Ib Trial of Copanlisib, A Phosphoinositide-3 Kinase (PI3K) Inhibitor, with Trastuzumab in Advanced Pre-Treated HER2-Positive Breast Cancer "PantHER".

BACKGROUND: Activation of the phosphoinositide-3 kinase (PI3K) pathway is a resistance mechanism to anti-human epidermal growth factor receptor 2 (HER2) therapy. This phase Ib trial was conducted to determine the maximum tolerated dose (MTD) of copanlisib, an intravenous (IV) pan-class I PI3K inhibitor, combined with trastuzumab. METHODS: Patients with advanced HER2-positive breast cancer and disease progression following at least one prior line of HER2 therapy in the metastatic setting were treated with copanlisib (45 or 60 mg) IV on days 1, 8 and 15 of a 28-day cycle with a fixed dose of trastuzumab 2 mg/kg weekly. RESULTS: Twelve patients were enrolled. The MTD was determined as copanlisib 60 mg plus trastuzumab 2 mg/kg weekly. The most common adverse events of any grade occurring in more than two patients were hyperglycaemia (58%), fatigue (58%), nausea (58%) and hypertension (50%). Stable disease was confirmed at 16 weeks in six participants (50%). PIK3CA mutations were detected in archival tumour of six participants (50%). PIK3CA hotspot mutations, were detectable in pre- and on-treatment plasma of all participants. Pre- and post-treatment tumour biopsies for two patients identified temporal genomic heterogeneity, somatic mutations in the TRRAP gene, which encodes a PI3K-like protein kinase, and emergent somatic mutations related to protein kinase signalling. CONCLUSION: Copanlisib and trastuzumab can be safely administered with fair overall tolerability. Preliminary evidence of tumour stability was observed in patients with heavily pre-treated, metastatic HER2 positive breast cancer. Several potential biomarkers were identified for further study in the current phase 2 clinical trial. NCT: 02705859.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Analysis of the reaction coordinate of alpha-L-fucosidases: a combined structural and quantum mechanical approach.

The enzymatic hydrolysis of alpha-L-fucosides is of importance in cancer, bacterial infections, and fucosidosis, a neurodegenerative lysosomal storage disorder. Here we show a series of snapshots along the reaction coordinate of a glycoside hydrolase family GH29 alpha-L-fucosidase unveiling a Michaelis (ES) complex in a (1)C(4) (chair) conformation and a covalent glycosyl-enzyme intermediate in (3)S(1) (skew-boat). First principles metadynamics simulations on isolated alpha-L-fucose strongly support a (1)C(4)<-->(3)H(4)<-->(3)S(1) conformational itinerary for the glycosylation step of the reaction mechanism and indicate a strong "preactivation" of the (1)C(4) complex to nucleophilic attack as reflected by free energy, C1-O1/O5-C1 bond length elongation/reduction, C1-O1 bond orientation, and positive charge development around the anomeric carbon. Analysis of an imino sugar inhibitor is consistent with tight binding of a chair-conformed charged species.

Agrobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis.

Agrobacterium tumefaciens VirB10 couples inner membrane (IM) ATP energy consumption to substrate transfer through the VirB/D4 type IV secretion (T4S) channel and also mediates biogenesis of the virB-encoded T pilus. Here, we determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) alpha-helix, (iii) proline-rich region (PRR) and (iv) C-terminal beta-barrel domain. Mutations conferring a transfer- and pilus-minus (Tra(-), Pil(-)) phenotype included PRR deletion and beta-barrel substitution mutations that prevented VirB10 interaction with the outer membrane (OM) VirB7-VirB9 channel complex. Mutations permissive for substrate transfer but blocking pilus production (Tra(+), Pil(-)) included a cytoplasmic domain deletion and TM domain insertion mutations. Another class of Tra(+) mutations also selectively disrupted pilus biogenesis but caused release of pilin monomers to the milieu; these mutations included deletions of alpha-helical projections extending from the beta-barrel domain. Our findings, together with results of Cys accessibility studies, indicate that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its beta-barrel domain with the VirB7-VirB9 channel complex. The data further support a model that distinct domains of VirB10 regulate formation of the secretion channel or the T pilus.

2-Phenyl-9H-purine-6-carbonitrile derivatives as selective cathepsin S inhibitors.

Starting from previously disclosed equally potent cathepsin K and S inhibitor 4-propyl-6-(3-trifluoromethylphenyl)pyrimidine-2-carbonitrile 1, a novel 2-phenyl-9H-purine-6-carbonitrile scaffold was identified to provide potent and selective cathepsin S inhibitors.

Trifluoromethylphenyl as P2 for ketoamide-based cathepsin S inhibitors.

The trifluoromethylphenyl P2 motif from previously reported heteroarylnitrile series has been successfully applied for the design and synthesis of highly potent novel ketoamide-based cathepsin S inhibitors. The key in this process is the change of the torsion angle between the P2 phenyl ring and the attached secondary amide by adding a small Cl, F, or Me group at the 2-position.

Design and optimization of a series of novel 2-cyano-pyrimidines as cathepsin K inhibitors.

Morphing structural features of HTS-derived chemotypes led to the discovery of novel 2-cyano-pyrimidine inhibitors of cathepsin K with good pharmacokinetic profiles, for example, compound 20 showed high catK potency (IC(50)=4nM), >580-fold selectivity over catL and catB, and oral bioavailability in the rat of 52%.

Optimisation of 2-cyano-pyrimidine inhibitors of cathepsin K: improving selectivity over hERG.

Several structure-guided optimisation strategies were explored in order to improve the hERG selectivity profile of cathepsin K inhibitor 1, whilst maintaining its otherwise excellent in vitro and in vivo profile. Ultimately, attenuation of clogP and pK(a) properties proved a successful approach and led to the discovery of a potent analogue 23, which, in addition to the desired selectivity over hERG (>1000-fold), displayed a highly attractive overall profile.

Tuned methods for conjugate addition to a vinyl oxadiazole; synthesis of pharmaceutically important motifs.

The addition of various nucleophiles to a vinyl 1,2,4-oxadiazole is described. Following optimisation, individual protocols tuned for the use of each specific class of reagent have been developed to allow the installation of nitrogen, sulfur, oxygen, and carbon nucleophiles, and leading to the preparation of a series of compounds containing the pharmaceutically important oxadiazole motif.

Extension of hepatic necrosis secondary to current arcing to surgical clips: a potential complication of radiofrequency ablation.

The authors present a case report of a 67-year-old woman who underwent radiofrequency ablation of recurrent hepatic metastases. She was managed 2 years previously with a right hemi-hepatectomy. Subsequent to RF ablation she developed hepatic necrosis extending in a linear fashion to two of the metallic surgical clips at the free edge of the liver, consistent with current arcing.

Small bowel imaging: CT enteroclysis or barium enteroclysis? Critically appraised topic.

Barium enteroclysis has been traditionally used in the diagnosis of small bowel Crohn's disease. Recently CT enteroclysis has been developed as an alternative imaging technique for small bowel Crohn's disease. A search and critical appraisal of the literature was performed to determine which technique is better for diagnosis of Crohn's disease. The best current evidence indicates that CT enteroclysis is a good test for the diagnosis of Crohn's disease but barium enteroclysis may also be required in a small group of patients.

MRI features after radiofrequency ablation of osteoid osteoma with cooled probes and impedance-control energy delivery.

OBJECTIVE: The purposes of our study were to determine the temporal changes in MR signal in bone after radiofrequency ablation of osteoid osteoma and the size of the zone of marrow signal change produced by the radiofrequency technique and to compare the size of the zone with published data for radiofrequency ablation with manual-control protocols. MATERIALS AND METHODS: Radiofrequency ablation was performed in 10 patients with a clinical and radiologic diagnosis of osteoid osteoma. A cooled radiofrequency probe was inserted in the nidus. Twelve minutes of radiofrequency energy was applied from a 200-W radiofrequency generator in an impedance-control setting. MRI with multiplanar turbo spin-echo T1-weighted and STIR sequences was performed at 1, 7, and 28 days after the procedure in seven patients. The three remaining patients had follow-up imaging at 28 days only. The images were reviewed by two radiologists who categorized the imaging features and measured the marrow zone of signal alteration when visible. The size of the zone of marrow signal change produced by the radiofrequency technique was compared with published data for radiofrequency ablation with manual-control protocols. RESULTS: A 1-mm band of homogeneous altered marrow signal distributed symmetrically parallel to the entire probe tract was seen earliest, at 1 day, in the femoral neck lesion treated with the 2-cm probe. The band was low signal on the T1 sequence and high signal on the STIR sequence, and the diameter of the zone was 27 mm. By 7 days, five of the seven treated bones showed a band of marrow signal alteration. By 28 days, all 10 treated bones had a band of marrow signal alteration. The interband distance at 90 degrees to the probe measured on STIR images at 28 days was a mean of 20.9 mm (confidence interval, 16.1-25.7 mm [p < 0.05]; range +/- measurement error, 10.5-35 +/- 1.64 mm) with a 1-cm probe and 30.5 mm (measurement error, +/- 0.78 mm) on T1 images without contrast material when a 2-cm exposed-tip probe was used. Higher-output generators with impedance-control software and internally cooled radiofrequency probes with longer exposed tips produce larger zones of marrow signal change than expected with manual-control protocols. CONCLUSION: MRI allows detection of temporal marrow signal change after radiofrequency ablation. The marrow signal change with a high-energy delivery protocol is larger than manual-control protocols.

Roles of the lactogens and somatogens in perinatal and postnatal metabolism and growth: studies of a novel mouse model combining lactogen resistance and growth hormone deficiency.

To delineate the roles of the lactogens and GH in the control of perinatal and postnatal growth, fat deposition, insulin production, and insulin action, we generated a novel mouse model that combines resistance to all lactogenic hormones with a severe deficiency of pituitary GH. The model was created by breeding PRL receptor (PRLR)-deficient (knockout) males with GH-deficient (little) females. In contrast to mice with isolated GH or PRLR deficiencies, double-mutant (lactogen-resistant and GH-deficient) mice on d 7 of life had growth failure and hypoglycemia. These findings suggest that lactogens and GH act in concert to facilitate weight gain and glucose homeostasis during the perinatal period. Plasma insulin and IGF-I and IGF-II concentrations were decreased in both GH-deficient and double-mutant neonates but were normal in PRLR-deficient mice. Body weights of the double mutants were reduced markedly during the first 3-4 months of age, and adults had striking reductions in femur length, plasma IGF-I and IGF binding protein-3 concentrations, and femoral bone mineral density. By age 6-12 months, however, the double-mutant mice developed obesity, hyperleptinemia, fasting hyperglycemia, relative hypoinsulinemia, insulin resistance, and glucose intolerance; males were affected to a greater degree than females. The combination of perinatal growth failure and late-onset obesity and insulin resistance suggests that the lactogen-resistant/GH-deficient mouse may serve as a model for the development of the metabolic syndrome.