Surface ID: a geometry-aware system for protein molecular surface comparison.

MOTIVATION: A protein can be represented in several forms, including its 1D sequence, 3D atom coordinates, and molecular surface. A protein surface contains rich structural and chemical features directly related to the protein's function such as its ability to interact with other molecules. While many methods have been developed for comparing the similarity of proteins using the sequence and structural representations, computational methods based on molecular surface representation are limited. RESULTS: Here, we describe "Surface ID," a geometric deep learning system for high-throughput surface comparison based on geometric and chemical features. Surface ID offers a novel grouping and alignment algorithm useful for clustering proteins by function, visualization, and in silico screening of potential binding partners to a target molecule. Our method demonstrates top performance in surface similarity assessment, indicating great potential for protein functional annotation, a major need in protein engineering and therapeutic design. AVAILABILITY AND IMPLEMENTATION: Source code for the Surface ID model, trained weights, and inference script are available at https://github.com/Sanofi-Public/LMR-SurfaceID.

A Greater Platelet Dose May Yield Better Clinical Outcomes for Platelet-Rich Plasma in the Treatment of Knee Osteoarthritis: A Systematic Review.

PURPOSE: To determine whether the platelet dose administered during a platelet-rich plasma (PRP) injection for knee osteoarthritis (OA) affects clinical outcomes. METHODS: A systematic review was performed by searching PubMed, Cochrane Library, and Embase for randomized controlled trials with at least 1 study arm using PRP for knee OA. Only studies that provided a platelet count, concentration, or dose with a minimum of 6-month outcome scores were included. Studies in which the PRP group had statistically significant positive outcomes were separated from those without statistical significance. The average platelet doses for studies with positive outcomes in the PRP group were compared with those without positive outcomes. RESULTS: After exclusion criteria were applied, 29 studies were analyzed. Of the 29, there were 31 arms that used PRP as a treatment method, of which 28 had statistically significant positive outcomes at 6 months compared with the control group. The mean platelet dose in the 28 with a positive outcome was 5,500 ± 474 × 106, whereas the 3 that had no positive difference had a mean platelet dose of 2,302 ± 437 × 106 (P < .01). There were 18 studies with 12-month outcomes, with 16 of 18 having positive outcomes. The positive studies had an average platelet dose of 5,464 ± 511, whereas the studies that had no statistical difference had an average platelet dose of 2,253 ± 753 × 106 (P < .05). CONCLUSIONS: Improved clinical outcomes from PRP injections for knee OA may be related to a greater platelet dose. LEVEL OF EVIDENCE: Level II, systematic review of Level I and II studies.

No Differences in 2-Year Reoperation Rates for Meniscus Allograft Transplant With Concomitant Cartilage Restoration or Osteotomy: A National Database Study.

PURPOSE: To investigate reoperation rates after meniscus allograft transplant (MAT), comparing rates with and without concomitant articular cartilage and osteotomy procedures using a national insurance claims database. METHODS: We performed a retrospective cohort study of patients who underwent MAT from 2010 to 2021 with minimum 2 year follow-up using the PearlDiver database. Using Current Procedural Terminology (CPT) and International Classification of Diseases (ICD) codes, we identified patients who underwent concomitant procedures including chondroplasty or microfracture, cartilage restoration defined as osteochondral graft or autologous chondrocyte implantation (ACI), or osteotomy. Univariate logistic regressions identified risk factors for reoperation. Reoperations were classified as knee arthroplasty, interventional procedures, or diagnostic or debridement procedures. RESULTS: 750 patients were included with an average age of 29.6 years (interquartile range 21.0-36.8) and average follow-up time was 5.41 years (SD: 2.51). 90-day, 2-year, and all-time reoperation rates were 1.33%, 14.4%, and 27.6% respectively. MAT with cartilage restoration was associated with increased reoperation rate at 90 days (OR: 4.88; 95% CI: 1.38-19.27; p=.015), however there was no significant difference in reoperation rates at 2 years or to the end of follow-up. ACI had increased reoperation rates at 90 days (OR: 6.95; 95% CI: 1.45-25.96; p=.006), with no difference in reoperation rates 2 years post-operatively or to the end of follow-up. Osteochondral autograft and allograft were not associated with increased reoperation rates. CONCLUSION: 14.4% of patients in our cohort had a reoperation within 2 years of MAT. Nearly one in four patients undergoing MAT had concomitant cartilage restoration, showing that it is commonly performed on patients with articular cartilage damage. Concomitant osteochondral autograft, osteochondral allograft, chondroplasty, microfracture and osteotomy were not associated with any significant difference in reoperation rates. ACI was associated with increased reoperation rates at 90 days, but not later.

Mechanistic incorporation of FcRn binding in plasma and endosomes in a whole body PBPK model for large molecules.

Monoclonal antibodies, endogenous IgG, and serum albumin bind to FcRn in the endosome for salvaging and recycling after pinocytotic uptake, which prolongs their half-life. This mechanism has been broadly recognized and is incorporated in currently available PBPK models. Newer types of large molecules have been designed and developed, which also bind to FcRn in the plasma space for various mechanistic reasons. To incorporate FcRn binding affinity in PBPK models, binding in the plasma space and subsequent internalisation into the endosome needs to be explicitly represented. This study investigates the large molecules model in PK-Sim® and its applicability to molecules with FcRn binding affinity in plasma. With this purpose, simulations of biologicals with and without plasma binding to FcRn were performed with the large molecule model in PK-Sim®. Subsequently, this model was extended to ensure a more mechanistic description of the internalisation of FcRn and the FcRn-drug complexes. Finally, the newly developed model was used in simulations to explore the sensitivity for FcRn binding in the plasma space, and it was fitted to an in vivo dataset of wild-type IgG and FcRn inhibitor plasma concentrations in Tg32 mice. The extended model demonstrated a strongly increased sensitivity of the terminal half-life towards the plasma FcRn binding affinity and could successfully fit the in vivo dataset in Tg32 mice with meaningful parameter estimates.

Accuracy of the Breast Cancer Surveillance Consortium Model Among Women with LCIS.

PURPOSE: The Breast Cancer Surveillance Consortium (BCSC) model predicts risk of invasive breast cancer risk based on age, race, family history, breast density, and history of benign breast disease, including lobular carcinoma in situ (LCIS). However, validation studies for this model included few women with LCIS. We sought to evaluate the accuracy of the BCSC model among this cohort. METHODS: Women with LCIS diagnosed between 1983 and 2017 were identified from a prospectively maintained database. The BCSC score was calculated; those with prior breast cancer, unknown breast density, age < 35 years or > 74 years, or with history of chemoprevention use were excluded. The Kaplan-Meier method was used to estimate incidence rates. Time-dependent receiver operating characteristic (ROC) analysis was used to analyze the discriminative capacity of the model. RESULTS: 1302 women with LCIS were included. At a median follow-up of 7 years, 152 women (12%) developed invasive cancer (6 with bilateral disease). Cumulative incidences of invasive breast cancer were 7.1% (95% CI 5.6-8.7) and 13.3% (95% CI 10.9-15.6), respectively, and the median BCSC risk scores were 4.9 and 10.4, respectively, at 5 and 10 years. The median 10-year BCSC score was significantly lower than the 10-year Tyrer-Cuzick score (10.4 vs 20.8, p < 0.001). The ROC curve scores (AUC) for BCSC at 5 and 10 years were 0.59 (95% CI 0.52-0.66) and 0.58 (95% CI 0.52-0.64), respectively. CONCLUSION: The BCSC model has moderate accuracy in predicting invasive breast cancer risk among women with LCIS with fair discrimination for risk prediction between individuals.

Synchronous and metachronous bilateral breast cancer among women with a history of lobular carcinoma in situ.

PURPOSE: Lobular carcinoma in situ (LCIS) confers increased cancer risk in either breast, but it remains unclear if this population is at increased risk for bilateral breast cancer (BC) development. Here we report bilateral BC incidence among women with a history of LCIS. METHODS: Women with classic-type LCIS diagnosed from 1980 to 2017 who developed unilateral BC (UBC) or bilateral BC were identified. Bilateral BC was categorized as synchronous (bilateral BC diagnosed < 6 months apart; SBBC) or metachronous (bilateral BC diagnosed ≥ 6 months apart; MBBC). Five-year incidence rates of bilateral BC among this population were evaluated. Comparisons were made to identify factors associated with bilateral BC. RESULTS: At 7 years' median follow-up, 249/1651 (15%) women with LCIS developed BC; 34 with bilateral BC (2%). There were no clinicopathologic feature differences between those with UBC and bilateral BC. SBBC occurred in 18 without significant differences versus UBC. Among 211 with UBC and a contralateral breast at risk, 16 developed MBBC at a median follow-up of 3 years. MBBC patients were less likely to receive endocrine therapy and more likely to receive chemotherapy versus UBC. Tumor histology was not associated with MBBC. Estimated 5-year MBBC risk was 6.4%. Index estrogen/progesterone receptor positivity and endocrine therapy were the only factors associated with MBBC risk. CONCLUSION: Bilateral BC occurred in 2% of women with LCIS history at median follow-up of 7 years. Similar to the general BC population, a decrease in MBBC is seen among women with a history of LCIS who develop hormone receptor-positive disease and those who receive endocrine therapy, highlighting the protective effects of this treatment.

Comparison of Outcomes for Classic-Type Lobular Carcinoma In Situ Managed with Surgical Excision After Core Biopsy Versus Observation.

BACKGROUND: Studies report low upgrade rates following excision for classic-type lobular carcinoma in situ (LCIS) with radiologic-pathologic concordance. Thus, in the absence of other high-risk lesions, observation has become standard. We report long-term outcomes of excision versus observation following a core biopsy diagnosis of classic-type LCIS. METHODS: Women with LCIS treated from 2013-2020 and managed with excision or observation were identified from a prospective database. Women with cancer upgrade at excision or history of cancer were excluded. We compared rates and characteristics of subsequent breast cancers by clinical management strategy. RESULTS: Of 312 women, 170 (54%) underwent excision and 142 (46%) were managed with observation. Among the excision group, 36 of 170 (21%) had radiologic-pathologic concordant LCIS without other high-risk lesions, mass, or symptoms (concordant LCIS excision group). Overall, at 3.1 years median follow-up, 11 (6.5%) women managed with excision and 11 (7.7%) women managed with observation developed cancer. Cancer development was not associated with management choice (overall excision cohort vs. observation group [p = 0.8]) and did not differ between the concordant LCIS excision and observation groups (p > 0.9). The 5-year cancer development rate was 8.9% (95% confidence interval [CI]: 2.3-31.6%) in the concordant LCIS excision group and 10.3% (95% CI 5.5-18.6%) in the observation group. CONCLUSIONS: No difference in breast cancer rates existed among women with a core-biopsy diagnosis of classic-type LCIS managed with excision or observation. These data support management of LCIS as a risk factor, with consideration of chemoprophylaxis, rather than as an indication for surgical excision.

Mapping of a Novel H3-Specific Broadly Neutralizing Monoclonal Antibody Targeting the Hemagglutinin Globular Head Isolated from an Elite Influenza Virus-Immunized Donor Exhibiting Serological Breadth.

The discovery of potent and broadly protective influenza virus epitopes could lead to improved vaccines that are resistant to antigenic drift. Here, we describe human antibody C585, isolated from a vaccinee with remarkable serological breadth as measured by hemagglutinin inhibition (HAI). C585 binds and neutralizes multiple H3N2 strains isolated between 1968 and 2016, including strains that emerged up to 4 years after B cells were isolated from the vaccinated donor. The crystal structure of C585 Fab in complex with the HA from A/Switzerland/9715293/2013 (H3N2) shows that the antibody binds to a novel and well-conserved epitope on the globular head of H3 HA and that it differs from other antibodies not only in its epitope but in its binding geometry and hypermutated framework 3 region, thereby explaining its breadth and ability to mediate hemagglutination inhibition across decades of H3N2 strains. The existence of epitopes such as the one elucidated by C585 has implications for rational vaccine design.IMPORTANCE Influenza viruses escape immunity through continuous antigenic changes that occur predominantly on the viral hemagglutinin (HA). Induction of broadly neutralizing antibodies (bnAbs) targeting conserved epitopes following vaccination is a goal of universal influenza vaccines and advantageous in protecting hosts against virus evolution and antigenic drift. To date, most of the discovered bnAbs bind either to conserved sites in the stem region or to the sialic acid-binding pocket. Generally, antibodies targeting the stem region offer broader breadth with low potency, while antibodies targeting the sialic acid-binding pocket cover narrower breadth but usually have higher potency. In this study, we identified a novel neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against a broad range of H3N2 with high potency. This epitope may provide insights for future universal vaccine design.

Mitochondrial Transplantation as a Novel Therapeutic Strategy for Mitochondrial Diseases.

Mitochondria are the major source of intercellular bioenergy in the form of ATP. They are necessary for cell survival and play many essential roles such as maintaining calcium homeostasis, body temperature, regulation of metabolism and apoptosis. Mitochondrial dysfunction has been observed in variety of diseases such as cardiovascular disease, aging, type 2 diabetes, cancer and degenerative brain disease. In other words, the interpretation and regulation of mitochondrial signals has the potential to be applied as a treatment for various diseases caused by mitochondrial disorders. In recent years, mitochondrial transplantation has increasingly been a topic of interest as an innovative strategy for the treatment of mitochondrial diseases by augmentation and replacement of mitochondria. In this review, we focus on diseases that are associated with mitochondrial dysfunction and highlight studies related to the rescue of tissue-specific mitochondrial disorders. We firmly believe that mitochondrial transplantation is an optimistic therapeutic approach in finding a potentially valuable treatment for a variety of mitochondrial diseases.

The Tyrer-Cuzick Model Inaccurately Predicts Invasive Breast Cancer Risk in Women With LCIS.

BACKGROUND: The Tyrer-Cuzick model has been shown to overestimate risk in women with atypical hyperplasia, although its accuracy among women with lobular carcinoma in situ (LCIS) is unknown. We evaluated the accuracy of the Tyrer-Cuzick model for predicting invasive breast cancer (IBC) development among women with LCIS. METHODS: Women with LCIS participating in surveillance from 1987 to 2017 were identified from a prospectively maintained database. Tyrer-Cuzick score (version 7) was calculated near the time of LCIS diagnosis. Patients with prior or concurrent breast cancer, a BRCA mutation, receiving chemoprevention, or with pleomorphic LCIS were excluded. Invasive cancer-free probability was estimated using the Kaplan-Meier method. RESULTS: A total of 1192 women with a median follow-up of 6 years (interquartile range [IQR] 2.5-9.9) were included. Median age at LCIS diagnosis was 49 years (IQR 45-55), 88% were white; 37% were postmenopausal, 28% had ≥ 1 first-degree family member with breast cancer, and 13% had ≥ 2 second-degree family members with breast cancer. In total, 128 patients developed an IBC; median age at diagnosis was 54 years (IQR 49-61). Five- and 10-year cumulative incidences of invasive cancer were 8% (95% confidence interval [CI] 6-9%) and 14% (95% CI 12-17%), respectively. The median Tyrer-Cuzick 10-year risk score was 20.1 (IQR 17.4-24.3). Discrimination measured by the C-index was 0.493, confirming that the Tyrer-Cuzick model is not well calibrated in this patient population. CONCLUSIONS: The Tyrer-Cuzick model is not accurate and may overpredict IBC risk for women with LCIS, and therefore should not be used for breast cancer risk assessment in this high-risk population.

Do Body Mass Index and Breast Density Impact Cancer Risk Among Women with Lobular Carcinoma In Situ?

PURPOSE: Both body mass index (BMI) and breast density impact breast cancer risk in the general population. Whether obesity and density represent additive risk factors in women with lobular carcinoma in situ (LCIS) is unknown. METHODS: Patients diagnosed with LCIS from 1988 to 2017 were identified from a prospectively maintained database. BMI was categorized by World Health Organization classification. Density was captured as the mammographic Breast Imaging Reporting and Data System (BIRADS) value. Other covariates included age at LCIS diagnosis, menopausal status, family history, chemoprevention, and prophylactic mastectomy. Cancer-free probability was estimated using the Kaplan-Meier method, and Cox regression models were used for univariable and multivariable analyses. RESULTS: A total of 1222 women with LCIS were identified. At a median follow-up of 7 years, 179 women developed breast cancer (121 invasive, 58 ductal carcinoma in situ); 5- and 10-year cumulative incidences of breast cancer were 10% and 17%, respectively. In multivariable analysis, increased breast density (BIRADS C/D vs. A/B) was significantly associated with increased hazard of breast cancer (hazard ratio [HR] 2.42, 95% confidence interval [CI] 1.52-3.88), whereas BMI was not. On multivariable analysis, chemoprevention use was associated with a significantly decreased hazard of breast cancer (HR 0.49, 95% CI 0.29-0.84). Exploratory analyses did not demonstrate significant interaction between BMI and menopausal status, BMI and breast density, BMI and chemoprevention use, or breast density and chemoprevention. CONCLUSIONS: Breast cancer risk among women with LCIS is impacted by breast density. These results aid in personalizing risk assessment among women with LCIS and highlight the importance of chemoprevention counseling for risk reduction.

Side Streets and U-Turns: Effects of Context Switching, Direction Switching, and Factor Switching on Interitem Correlations and Misresponse Rates.

This study tested implications of the context switching perspective proposed by Hamby, Ickes, and Babcock ( 2016 ). Using trained raters to assess the amount of reframing required to interpret the meaning of the subsequent (second) item within all adjacent item pairs, we first established that this process variable could be measured reliably. Then, in the data for 18 personality measures drawn from 3 individual-difference domains, we found that the amount of reframing (i.e., context switching) needed to interpret successive items predicted both lower interitem correlations and a greater percentage of misresponders. Similarly, item pairs that were mismatched in "directional" wording also predicted both lower interitem correlations and more misresponders. Finally, item pairs representing different factors predicted lower interitem correlations. Although the effects of direction switching and factor switching were partially mediated by the amount of reframing required, they remained significant even when the mediating effect of reframing was statistically controlled. These results indicate that interpreting the meaning of test items is a task for which the level of difficulty can vary with each successive item, as a function of how the current item compares to the previous item in aspects such as its context generality or specificity, directional wording, and content domain.

The Role of Adipose Tissue Mitochondria: Regulation of Mitochondrial Function for the Treatment of Metabolic Diseases.

: Mitochondria play a key role in maintaining energy homeostasis in metabolic tissues, including adipose tissues. The two main types of adipose tissues are the white adipose tissue (WAT) and the brown adipose tissue (BAT). WAT primarily stores excess energy, whereas BAT is predominantly responsible for energy expenditure by non-shivering thermogenesis through the mitochondria. WAT in response to appropriate stimuli such as cold exposure and ß-adrenergic agonist undergoes browning wherein it acts as BAT, which is characterized by the presence of a higher number of mitochondria. Mitochondrial dysfunction in adipocytes has been reported to have strong correlation with metabolic diseases, including obesity and type 2 diabetes. Dysfunction of mitochondria results in detrimental effects on adipocyte differentiation, lipid metabolism, insulin sensitivity, oxidative capacity, and thermogenesis, which consequently lead to metabolic diseases. Recent studies have shown that mitochondrial function can be improved by using thiazolidinedione, mitochondria-targeted antioxidants, and dietary natural compounds; by performing exercise; and by controlling caloric restriction, thereby maintaining the metabolic homeostasis by inducing adaptive thermogenesis of BAT and browning of WAT. In this review, we focus on and summarize the molecular regulation involved in the improvement of mitochondrial function in adipose tissues so that strategies can be developed to treat metabolic diseases.

A Toolbox for Rapid Quantitative Assessment of Chronological Lifespan and Survival in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a well-established model organism to study the mechanisms of longevity. One of the two aging paradigms studied in yeast is termed chronological lifespan (CLS). CLS is defined by the amount of time non-dividing yeast cells can survive at stationary phase. Here, we propose new approaches that allow rapid and efficient quantification of survival rates in aging yeast cultures using either a fluorescent cell counter or microplate imaging. We have generated a software called analysr (Analytical Algorithm for Yeast Survival Rates) that allows automated and highly reproducible analysis of cell survival in aging yeast cultures using fluorescent data. To demonstrate the efficiency of our new experimental tools, we tested the previously characterized ability of caloric restriction to extend lifespan. Interestingly, we found that this process is independent of the expression of three central yeast heat shock proteins (Hsp26, Hsp42, Hsp104). Finally, our new assay is easily adaptable to other types of toxicity studies. Here, we assessed the toxicity of various concentrations of acetic acid, a known contributor of yeast chronological aging. These assays provide researchers with cost-effective, low- and high-content assays that can serve as an efficient complement to the time-consuming colony forming unit assay usually used in CLS studies.

Cutting edge: cell surface linker for activation of T cells is recruited to microclusters and is active in signaling.

A controversy has recently emerged regarding the location of the cellular pool of the adapter linker for activation of T cells (LAT) that participates in propagation of signals downstream of the TCR. In one model phosphorylation and direct recruitment of cell surface LAT to activation-induced microclusters is critical for T cell activation, whereas in the other model vesicular, but not surface, LAT participates in these processes. By using a chimeric version of LAT that can be tracked via an extracellular domain, we provide evidence that LAT located at the cell surface can be recruited efficiently to activation-induced microclusters within seconds of TCR engagement. Importantly, we also demonstrate that this pool of LAT at the plasma membrane is rapidly phosphorylated. Our results provide support for the model in which the cell utilizes LAT from the cell surface for rapid responses to TCR stimulation.

Essential role of the cancer stem/progenitor cell marker nucleostemin for indole-3-carbinol anti-proliferative responsiveness in human breast cancer cells.

BACKGROUND: Nucleostemin is a nucleolus residing GTPase that is considered to be an important cancer stem/progenitor cell marker protein due to its high expression levels in breast cancer stem cells and its role in tumor-initiation of human mammary tumor cells. It has been proposed that nucleostemin may represent a valuable therapeutic target for breast cancer; however, to date evidence supporting the cellular mechanism has not been elucidated. RESULTS: Expression of exogenous HER2, a member of the EGF receptor gene family, in the human MCF-10AT preneoplastic mammary epithelial cell line formed a new breast cancer cell line, 10AT-Her2, which is highly enriched in cells with stem/progenitor cell-like character. 10AT-Her2 cells display a CD44+/CD24-/low phenotype with high levels of the cancer stem/progenitor cell marker proteins nucleostemin, and active aldehyde dehydrogenase-1. The overall expression pattern of HER2 protein and the stem/progenitor cell marker proteins in the 10AT-Her2 cell population is similar to that of the luminal HER2+ SKBR3 human breast cancer cell line, whereas, both MCF-7 and MDA-MB-231 cells display reduced levels of nucleostemin and no detectable expression of ALDH-1. Importantly, in contrast to the other well-established human breast cancer cell lines, 10AT-Her2 cells efficiently form tumorspheres in suspension cultures and initiate tumor xenograft formation in athymic mice at low cell numbers. Furthermore, 10AT-Her2 cells are highly sensitive to the anti-proliferative apoptotic effects of indole-3-carbinol (I3C), a natural anti-cancer indolecarbinol from cruciferous vegetables of the Brassica genus such as broccoli and cabbage. I3C promotes the interaction of nucleostemin with MDM2 (Murine Double Mutant 2), an inhibitor of the p53 tumor suppressor, and disrupts the MDM2 interaction with p53. I3C also induced nucleostemin to sequester MDM2 in a nucleolus compartment, thereby freeing p53 to mediate its apoptotic activity. siRNA knockdown of nucleostemin functionally documented that nucleostemin is required for I3C to trigger its cellular anti-proliferative responses, inhibit tumorsphere formation, and disrupt MDM2-p53 protein-protein interactions. Furthermore, expression of an I3C-resistant form of elastase, the only known target protein for I3C, prevented I3C anti-proliferative responses in cells and in tumor xenografts in vivo, as well as disrupt the I3C stimulated nucleostemin-MDM2 interactions. CONCLUSIONS: Our results provide the first evidence that a natural anti-cancer compound mediates its cellular and in vivo tumor anti-proliferative responses by selectively stimulating cellular interactions of the stem/progenitor cell marker nucleostemin with MDM2, which frees p53 to trigger its apoptotic response. Furthermore, our study provides a new mechanistic template that can be potentially exploited for the development of cancer stem/progenitor cell targeted therapeutic strategies.

Antioxidant properties of fermented mango leaf extracts.

Antioxidant properties of mango (Mangifera indica) leaves were evaluated. Hydroalcoholic leaf extracts that were lyophilized were subsequently fermented with either Lactobacillus casei or effective microorganisms (EM) such as probiotic bacteria and/or other anaerobic organisms. Antioxidant properties were measured as a function of the mango leaf extract concentration in the fermentation broth. Tests for radical scavenging using the 1,1-diphenyl-2-picrylhydrazyl radical showed higher antioxidant activity for Lactobacillus- and EM-fermented mango leaf extracts than for the synthetic antioxidant butylated hydroxytoluene. Antioxidant activity generally increased with increasing fermented extract concentration as did the fermented extracts' polyphenol and flavonoid contents. Fermented extracts reduced reactive oxygen species generation by lipopolysaccharide in RAW 264.7 cells when measured via fluorescence of dichlorodihydrofluorescein acetate treated cells using flow cytometry. RAW 264.7 cells also showed a concentration-dependent cytotoxic effect of the fermented extracts using the 3-(4,5-dimethylthialol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Inhibition of mushroom tyrosinase activity as well as nitrite scavenging by the fermented extracts increased as fermented extract concentrations increased. Tyrosinase activity was assayed with 3,4-dihydroxyphenylalanine as substrate. Nitrite scavenging was assessed via measurement of inhibition of chromophore production from nitrite-naphthylamine-sulfanilic acid mixtures. The antioxidant properties of fermented mango leaf extracts suggest the fermented extracts may be useful in developing health food and fermentation-based beauty products.

Amyloid-ß aggregation with gold nanoparticles on brain lipid bilayer.

Understanding and manipulating amyloid-ß (Aß) aggregation provide key knowledge and means for the diagnosis and cure of Alzheimer's disease (AD) and the applications of Aß-based aggregation systems. Here, we studied the formation of various Aß aggregate structures with gold nanoparticles (AuNPs) and brain total lipid extract-based supported lipid bilayer (brain SLB). The roles of AuNPs and brain SLB in forming Aß aggregates were studied in real time, and the structural details of Aß aggregates were monitored and analyzed with the dark-field imaging of plasmonic AuNPs that allows for long-term in situ imaging of Aß aggregates with great structural details without further labeling. It was shown that the fluid brain SLB platform provides the binding sites for Aß and drives the fast and efficient formation of Aß aggregate structures and, importantly, large Aß plaque structures (>15 µm in diameter), a hallmark for AD, were formed without going through fibril structures when Aß peptides were co-incubated with AuNPs on the brain SLB. The dark-field scattering and circular dichroism-correlation data suggest that AuNPs were heavily involved with Aß aggregation on the brain SLB and less α-helix, less ß-sheet and more random coil structures were found in large plaque-like Aß aggregates.

Site-specific antibody-drug conjugation through glycoengineering.

Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.

Targeted disruption of Tbc1d20 with zinc-finger nucleases causes cataracts and testicular abnormalities in mice.

BACKGROUND: Loss-of-function mutations in TBC1D20 cause Warburg Micro syndrome 4 (WARBM4), which is an autosomal recessive syndromic disorder characterized by eye, brain, and genital abnormalities. Blind sterile (bs) mice carry a Tbc1d20-null mutation and exhibit cataracts and testicular phenotypes similar to those observed in WARBM4 patients. In addition to TBC1D20, mutations in RAB3GAP1, RAB3GAP2 and RAB18 cause WARBM1-3 respectively. However, regardless of which gene harbors the causative mutation, all individuals affected with WARBM exhibit indistinguishable clinical presentations. In contrast, bs, Rab3gap1 (-/-) , and Rab18 (-/-) mice exhibit distinct phenotypes; this phenotypic variability of WARBM mice was previously attributed to potential compensatory mechanisms. Rab3gap1 (-/-) and Rab18 (-/-) mice were genetically engineered using standard approaches, whereas the Tbc1d20 mutation in the bs mice arose spontaneously. There is the possibility that another unidentified mutation within the bs linkage disequilibrium may be contributing to the bs phenotypes and thus contributing to the phenotypic variability in WARBM mice. The goal of this study was to establish the phenotypic consequences in mice caused by the disruption of the Tbc1d20 gene. RESULTS: The zinc finger nuclease (ZFN) mediated genomic editing generated a Tbc1d20 c.[418_426del] deletion encoding a putative TBC1D20-ZFN protein with an in-frame p.[H140_Y143del] deletion within the highly conserved TBC domain. The evaluation of Tbc1d20 (ZFN/ZFN) eyes identified severe cataracts and thickened pupillary sphincter muscle. Tbc1d20 (ZFN/ZFN) males are infertile and the analysis of the seminiferous tubules identified disrupted acrosomal development. The compound heterozygote Tbc1d20 (ZFN/bs) mice, generated from an allelic bs/+ X Tbc1d20 (ZFN/+) cross, exhibited cataracts and aberrant acrosomal development indicating a failure to complement. CONCLUSIONS: Our findings show that the disruption of Tbc1d20 in mice results in cataracts and aberrant acrosomal formation, thus establishing bs and Tbc1d20 (ZFN/ZFN) as allelic variants. Although the WARBM molecular disease etiology remains unclear, both the bs and Tbc1d20 (ZFN/ZFN) mice are excellent model organisms for future studies to establish TBC1D20-mediated molecular and cellular functions.