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A primary difference between black women (BW) and white women (WW) diagnosed with breast cancer is not incidence, but aggressiveness of the tumor. Black women have higher mortalities with similar incidence of breast cancer compared to other race/ethnicities, and are diagnosed at a younger age with more advanced tumors with double the rate of lethal, triple negative breast cancers. There is ongoing debate regarding whether the underlying cause of higher mortality is related to healthcare inequalities or due to ancestry dependent molecular features found in normal breast tissue that facilitate the aggressive phenotype found in black women. One hypothesis is that chronic social and economic stressors result in molecular responses that create a tumor permissive tissue microenvironment in normal breast tissue, and these chronic stresses differ by race/ethnicity. In this study, we investigate molecular pattern changes in tissue N-glycosylation in a cohort of ancestry defined normal breast tissue from BW and WW with significant 5-year risk of breast cancer by Gail score. N-glycosylation, a glucose metabolism-linked post-translational modification attached to an asparagine (N) residue, was tested against social stressors. Social stresses included marital status, single, education, economic status (income), personal reproductive history, the risk factors BMI and age. Normal breast tissue microarrays from the Susan G. Komen tissue bank (WB=43; WW= 43) were used to evaluate glycosylation against socioeconomic stress and risk factors. There was no significant difference in age (Median age BW 42.5, 95% CI [40.0, 45.0]; WW 42.0, 95% CI [39.7, 44.3]). A subgroup of women had similar BMI (BW, n=24 Median BMI 29.5, 95% CI [27.6, 32.3]; WW, n=24 28.3, 95% CI [23.5, 31.6]. BW women had an overall lower median risk by 5-year Gail score, which, (BW 9.4, 95% CI [8.9, 9.9]; WW 10.4, 95% CI [8.1, 12.6]). Area under the receiver operating curve (AUC) ≥0.70, Brown/Wilson p-value Citation Format: Peggi M Angel, Yeonhee Park, Danielle A Scott, Denys Rujchanarong, Sean Brown, Richard R Drake, George E Sandusky, Harikrishna Nakshatri. Metabolic links to socioeconomic stresses uniquely affecting race in normal breast tissue at risk for breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-13.

Our group has recently developed the GlycoTyper assay which is a streamlined antibody capture slide array approach to directly profile N-glycans of captured serum glycoproteins including immunoglobulin G (IgG). This method needs only a few microliters of serum and utilizes a simplified processing protocol that requires no purification or sugar modifications prior to analysis. In this method, antibody captured glycoproteins are treated with peptide N-glycosidase F (PNGase F) to release N-glycans for detection by MALDI imaging mass spectrometry (IMS). As alterations in N-linked glycans have been reported for IgG from large patient cohorts with fibrosis and cirrhosis, we utilized this novel method to examine the glycosylation of total IgG, as well as IgG1, IgG2, IgG3 and IgG4, which have never been examined before, in a cohort of 106 patients with biopsy confirmed liver fibrosis. Patients were classified as either having no evidence of fibrosis (41 patients with no liver disease or stage 0 fibrosis), early stage fibrosis (10 METAVIR stage 1 and 18 METAVIR stage 2) or late stage fibrosis (6 patients with METAVIR stage 3 fibrosis and 37 patients with METAVIR stage 4 fibrosis (cirrhosis)). Several major alterations in glycosylation were observed that classify patients as having no fibrosis (sensitivity of 92% and a specificity of 90%), early fibrosis (sensitivity of 84% with 90% specificity) or significant fibrosis (sensitivity of 94% with 90% specificity).

Introduction: For decades, the role of glycans and glycoproteins in the progression of breast cancer and other cancers have been evaluated. Through extensive studies focused on elucidating the biological functions of glycosylation, researchers have been able to implicate alterations in these functions to tumor formation and metastasis. Areas covered: In this review, we summarize how changes in glycosylation are associated with tumorigenesis, with emphasis on breast cancers. An overview of the changes in N-linked and O-linked glycans associated with breast cancer tumors and biofluids are described. Recent advances in glycomics are emphasized in the context of continuing to decipher the glycosylation changes associated with breast cancer progression. Expert opinion: While changes in glycosylation have been studied in breast cancer for many years, the clinical relevance of these studies has been limited. This reflects the inherent biological and clinical heterogeneity of breast cancers. Glycomics analysis lags behind the advances in genomics and proteomics, but new approaches are emerging. A summary of known glycosylation changes associated with breast cancer is necessary to implement new findings in the context of clinical outcomes and therapeutic strategies. A better understanding of the dynamics of tumor and immune glycosylation is critical to improving emerging immunotherapeutic treatments.

A number of inhibitors have been developed for the SARS‐CoV‐2 main protease (MPro) as potential COVID‐19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1‐9, GC376, 11a, 10–1, 10–2, and 10–3. MPI1‐9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10–1, 10–2 and 10–3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti‐SARS‐CoV‐2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS‐CoV‐2 cell entry. By selectively inhibiting both SARS‐CoV‐2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID‐19., Two targets, one goal: MPI8, a peptide aldehyde inhibitor of the SARS‐CoV‐2 main protease with high antiviral potency, also displays high potency in inhibiting human cathepsin L to exert potentially dual functions to inhibit the SARS‐CoV‐2 infection. This cathepsin L inhibition is selective over cathepsins B and K.

A number of inhibitors have been developed for the SARS-CoV-2 main protease (MPro) as potential COVID-19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1-9, GC376, 11a, 10-1, 10-2, and 10- 3. MPI1-9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10-1, 10-2 and 10-3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti-SARS-CoV-2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M (Pro) ) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M (Pro) ), we have designed and synthesized a series of SC2M (Pro) inhibitors that contain β-( S -2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M (Pro) active site cysteine C145. All inhibitors display high potency with IC (50) values at or below 100 nM. The most potent compound MPI3 has as an IC (50) value as 8.5 nM. Crystallographic analyses of SC2M (Pro) bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M (Pro) inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active site cysteine C145. All inhibitors display high potency with IC50 values at or below 100 nM. The most potent compound MPI3 has as an IC50 value as 8.5 nM. Crystallographic analyses of SC2MPro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.

Objectives: Antipsychotics are commonly initiated in the hospital for agitation and delirium and may be inappropriately continued upon floor transfer and at discharge. We sought to evaluate the magnitude of this issue within our health care system. Methods: We conducted a multicenter, retrospective cohort study within a 22-hospital health care system to evaluate the proportion of patients without identifiable psychiatric illness who received newly initiated inpatient antipsychotics and were then continued on an antipsychotic at hospital discharge. Results: Of 23 049 patients who received at least 1 in-hospital dose of haloperidol, olanzapine, quetiapine, risperidone, or ziprasidone, 8297 patients were included in the final analysis after applying exclusion for identifiable psychiatric illness or previous antipsychotic use. Ultimately, 334 patients (4%) were discharged with a new antipsychotic prescription. Patients receiving antipsychotics at discharge were more likely as an inpatient to receive quetiapine (77.2% vs 35.9%; odds ratio [OR]: 6.1, 95% confidence interval [CI]: 4.7-8.0; P < .001) and less likely to receive haloperidol (15% vs 47%; OR: 0.2, 95% CI: 0.14-0.27; P < .001) or olanzapine (16.2% vs 20.9%; OR: 0.73, 95% CI: 0.53-0.98; P < .04). Conclusions: Antipsychotics may be inappropriately continued in non-psychiatric patients at hospital discharge. Strategies to limit potentially unnecessary antipsychotics upon discharge should be evaluated.

The TMEM165 gene encodes for a multiple pass membrane protein localized in the Golgi that has been linked to congenital disorders of glycosylation. The TMEM165 protein is a putative ion transporter that regulates H+/Ca++/Mn++ homeostasis and pH in the Golgi. Previously, we identified TMEM165 as a potential biomarker for breast carcinoma in a glycoproteomic study using late stage invasive ductal carcinoma tissues with patient- matched adjacent normal tissues. The TMEM165 protein was not detected in non-malignant matched breast tissues and was detected in invasive ductal breast carcinoma tissues by mass spectrometry. Our hypothesis is that the TMEM165 protein confers a growth advantage to breast cancer. In this preliminary study we have investigated the expression of TMEM165 in earlier stage invasive ductal carcinoma and ductal carcinoma in situ cases. We created a CRISPR/Cas9 knockout of TMEM165 in the human invasive breast cancer cell line MDAMB231. Our results indicate that removal of TMEM165 in these cells results in a significant reduction of cell migration, tumor growth, and tumor vascularization in vivo. Furthermore, we find that TMEM165 expression alters the glycosylation of breast cancer cells and these changes promote the invasion and growth of breast cancer by altering the expression levels of key glycoproteins involved in regulation of the epithelial to mesenchymal transition such as E-cadherin. These studies illustrate new potential functions for this Golgi membrane protein in the control of breast cancer growth and invasion. 11;28

(1) Glycoproteins account for ~80% of proteins located at the cell surface and in the extracellular matrix. A growing body of evidence indicates that &alpha, L-fucose protein modifications contribute to breast cancer progression and metastatic disease. (2) Using a combination of techniques, including matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) based in cell and on tissue imaging and glycan sequencing using exoglycosidase analysis coupled to hydrophilic interaction ultra-high performance liquid chromatography (HILIC UPLC), we establish that a core-fucosylated tetra-antennary glycan containing a single N-acetyllactosamine (F(6)A4G4Lac1) is associated with poor clinical outcomes in breast cancer, including lymph node metastasis, recurrent disease, and reduced survival. (3) This study is the first to identify a single N-glycan, F(6)A4G4Lac1, as having a correlation with poor clinical outcomes in breast cancer.

Purpose Using a recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method, human breast cancer formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) are evaluated for N-linked glycan distribution in the tumor microenvironment. Experimental design Tissue sections representing multiple human epidermal growth factor receptor 2 (HER2) receptor-positive and triple-negative breast cancers (TNBC) in both TMA and FFPE slide format are processed for high resolution N-glycan MALDI-IMS. An additional FFPE tissue cohort of primary and metastatic breast tumors from the same donors are also evaluated. Results The cumulative N-glycan MALDI-IMS analysis of breast cancer FFPE tissues and TMAs indicate the distribution of specific glycan structural classes to stromal, necrotic, and tumor regions. A series of high-mannose, branched and fucosylated glycans are detected predominantly within tumor regions. Additionally, a series of polylactosamine glycans are detected in advanced HER2+, TNBC, and metastatic breast cancer tissues. Comparison of tumor N-glycan species detected in paired primary and metastatic tissues indicate minimal changes between the two conditions. Conclusions and clinical relevance The prevalence of tumor-associated polylactosamine glycans in primary and metastatic breast cancer tissues indicates new mechanistic insights into the development and progression of breast cancers. The presence of these glycans could be targeted for therapeutic strategies and further evaluation as potential prognostic biomarkers.

Tissue necrosis is a form of cell death common in advanced and aggressive solid tumors, and is associated with areas of intratumoral chronic ischemia. The histopathology of necrotic regions appear as a scaffold of cellular membrane remnants, reflective of the hypoxia and cell degradation events associated with this cellular death pathway. Changes in the glycosylation of cell surface proteins is another common feature of cancer progression. Using a recently developed mass spectrometry imaging approach to evaluate N-linked glycan distributions in human formalin-fixed clinical cancer tissues, differences in the glycan structures of regions of tumor, stroma and necrosis were evaluated. While the structural glycan classes detected in the tumor and stromal regions are typically classified as high mannose or branched glycans, the glycans found in necrotic regions displayed limited branching, contained sialic acid modifications and lack fucose modifications. While this phenomenon was initially classified in breast cancer tissues, it has been also seen in cervical, thyroid and liver cancer samples. These changes in glycosylation within the necrotic regions could provide further mechanistic insight to necrotic changes in cancer tissue and provide new research directions for identifying prognostic markers of necrosis.

In 2001, the US Food and Drug Administration approved recombinant tissue plasminogen activator (alteplase, Cathflo Activase) to reestablish patency of central catheters occluded, presumably, by a fibrin clot. We conducted a multicenter quality improvement study to determine the value of this procedure in our Neonatal Intensive Care Unit (NICUs), including analyses of efficacy, safety and costs.We conducted a retrospective quality analysis of neonates in level III NICUs, who received alteplase for the purpose of reestablishing patency of occluded central catheters.Alteplase was administered to 169 neonates, each given one to four doses, totaling 205 episodes of administration. The most common type of catheter where alteplase was used was percutaneously inserted central catheter (PICC) lines (78% of uses), 8% were umbilical venous catheters (UVCs), 6% arterial lines, 5% chest tubes and 3% other catheters. Postnatal age at first dose ranged from 0 to 132 days (median, 12); dosed patients were 22 to 41 weeks gestation at birth (median, 31). Fifty-eight percentage of administrations restored catheter function. Success was more likely at younger postnatal age (10±2 days old in successful vs 14±1 days in unsuccessful treatments; P=0.023). Seventy-two percentage of the re-canalized catheters remained functional until they were no longer needed (2 to 30 days later). Nine percentage of episodes were treated with a second dose 1 to 17 days later for re-occlusion and 50% of those were successful. Bleeding consequences were identified in only one case, where three separate lines were treated (chest tube, PICC and UVC) within a 6-h period. Costs to the health system of doses, minus savings to the system by not needing to replace lines, averaged a net of $34 per dose.The apparent safety and favorable value analysis prompted us to develop a consistent approach to alteplase usage in the Intermountain Healthcare NICUs, using the data in this report to standardize the guidelines across our health system.

Breast cancer characterized by human epidermal growth factor receptor 2 (HER2) overexpression represents approximately 25-30% of all breast cancer cases. Many patients acquire resistance to current chemotherapies, leading to a more aggressive disease state with severe clinical outcomes. Lapatinib, a first generation tyrosine kinase inhibitor of HER2 and EGFR (epidermal growth factor receptor), is commonly used to treat HER2+ breast cancer. Resistance to lapatinib is steadily increasing among HER2+ patients, highlighting the need for therapy development. Identifying markers that predict treatment response or potential drug targets could enhance treatment efficacy and patient survival. To investigate this, we have used MALDI mass spectrometry to identify N-linked glycans specific to human breast cancer cell lines with known resistance and sensitivity to lapatinib treatment, JIMT-1 (resistant) HER2+ and BT474 (sensitive) HER2+. After different lapatinib dose and time course experiments, N-linked glycans were isolated and comparatively profiled by high resolution MALDI mass spectrometry. Differences in the levels of fucosylation and sialylation of glycans from sensitive and resistant cell lines, before and after treatment, were evaluated. In addition, mouse xenograft tumor tissues derived from the same cell lines treated with and without lapatinib were processed for on-tissue imaging of N-glycans using a MALDI imaging mass spectrometry approach. Tissues from HER2+ human breast tumors were also imaged with the same MALDI imaging approach. Cumulatively, these preliminary studies have identified novel glycosylation patterns associated with lapatinib treatment sensitivity and resistance. Citation Format: Scott DA, Angel P, Xu H, Drake R, Yeh E. Identifying novel glycosylation markers in Her2+ breast cancer using MALDI mass spectrometry [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-30.

Professional societies have published recommendations for iron dosing of preterm neonates, but differences exist between guidelines. To help develop standardized guidelines, we performed a 10-year analysis of iron dosing in groups at risk for iron deficiency: IDM (infants of diabetic mothers), SGA (small for gestational age), and VLBW premature neonates (very low birth weight