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Calorie labeling is a recent initiative from the Saudi Food and Drug Authority (SFDA) aimed to reduce the prevalence of noncommunicable diseases (NCDs) by influencing people to make healthier food choices when they eat out and can also help people with weight disturbances to be more aware of their calorie intake. The present study aimed to investigate the association between the use of calorie labeling on restaurant menus, calorie intake, weight concern, body weight perception, and weight-control behaviors among young women. A quasi-experimental study was conducted among female students at a university restaurant. Participants were assigned to two groups: food menus with (experimental group) and without (control group) calorie labeling. The logistic regression model assessed the predictors of using calorie information separately for the experimental and control groups. Calorie labeling had a significant effect on reducing calorie consumption in the experimental group by 59 calories compared to the control group. The higher weight concern in the control group (OR = 0.410; 95% CI 0.230-0.730; P ≤ 0.002) was a predictor for using calorie information. The experimental group had higher weight concern (OR = 1.530; 95% CI 1.107-2.115; P ≤ 0.01) and body weight perception (OR = 4.230; 95% CI 1.084-6.517; P ≤ 0.038) and lower calorie intake (OR = 1.005; 95% CI 1.001-6.517; P ≤ 0.008) predictors for using calorie information. Weight-control behaviors did not significantly predict the use of calorie information in the groups. Calorie labeling might increase the weight disturbances among young females. More investigation is needed across various populations to gain a better understanding of calorie labeling as an effective food choice among people who are vulnerable to weight disturbances or already have weight disorders., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Hala Al-Otaibi et al.)

We describe the 'Crescendo Mouse', a human V H transgenic platform combining an engineered heavy chain locus with diverse human heavy chain V, D and J genes, a modified mouse Cγ1 gene and complete 3' regulatory region, in a triple knock-out (TKO) mouse background devoid of endogenous immunoglobulin expression. The addition of the engineered heavy chain locus to the TKO mouse restored B cell development, giving rise to functional B cells that responded to immunization with a diverse response that comprised entirely 'heavy chain only' antibodies. Heavy chain variable (V H ) domain libraries were rapidly mined using phage display technology, yielding diverse high-affinity human V H that had undergone somatic hypermutation, lacked aggregation and showed enhanced expression in E. coli. The Crescendo Mouse produces human V H fragments, or Humabody® V H , with excellent bio-therapeutic potential, as exemplified here by the generation of antagonistic Humabody® V H specific for human IL17A and IL17RA., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA-approved antibody-drug conjugates (ADC). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of prostate-specific membrane antigen-binding single-domain (Humabody) ADC constructs to demonstrate that tissue penetration of protein-drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin-binding domain to the single-domain ADCs. A construct lacking an albumin-binding domain was rapidly cleared, leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein-drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing. SIGNIFICANCE: A mechanistic study of protein-drug conjugates demonstrates that a lower potency compound is more effective in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution., (©2020 American Association for Cancer Research.)

Repurposing "old" drugs can facilitate rapid clinical translation but necessitates novel mechanistic insight. Warfarin, a vitamin K "antagonist" used clinically for the prevention of thrombosis for more than 50 years, has been shown to have anticancer effects. We hypothesized that the molecular mechanism underlying its antitumor activity is unrelated to its effect on coagulation, but is due to inhibition of the Axl receptor tyrosine kinase on tumor cells. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here, we show that inhibiting Gas6-dependent Axl activation with low-dose warfarin, or with other tumor-specific Axl-targeting agents, blocks the progression and spread of pancreatic cancer. Warfarin also inhibited Axl-dependent tumor cell migration, invasiveness, and proliferation while increasing apoptosis and sensitivity to chemotherapy. We conclude that Gas6-induced Axl signaling is a critical driver of pancreatic cancer progression and its inhibition with low-dose warfarin or other Axl-targeting agents may improve outcome in patients with Axl-expressing tumors., (©2015 American Association for Cancer Research.)

Background: To investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative) for pancreatic autoantibodies [islet cell autoantibodies(ICA), glutamic acid decarboxylase antibodies (GADA) and insulinoma-associated antigen-2 antibodies (IA-2A)]. Furthermore the study aimed at determining whether mutations in KCNJ11, ABCC8, HNF1A, HNF4A or INS are common in AAB negative diabetes., Materials and Methods: In 261 newly diagnosed children with type 1 diabetes, we measured residual β-cell function, ICA, GADA, and IA-2A at 1, 6 and 12 months after diagnosis. The genes KCNJ11, ABCC8, HNF1A, HNF4A and INS were sequenced in subjects AAB negative at diagnosis. We expressed recombinant K-ATP channels in Xenopus oocytes to analyse the functional effects of an ABCC8 mutation., Results: Twenty-four patients (9.1%) tested AAB negative after one month. Patients, who were AAB-negative throughout the 12-month period, had higher residual β-cell function (P = 0.002), lower blood glucose (P = 0.004), received less insulin (P = 0.05) and had lower HbA1c (P = 0.02) 12 months after diagnosis. One patient had a heterozygous mutation leading to the substitution of arginine at residue 1530 of SUR1 (ABCC8) by cysteine. Functional analyses of recombinant K-ATP channels showed that R1530C markedly reduced the sensitivity of the K-ATP channel to inhibition by MgATP. Morover, the channel was highly sensitive to sulphonylureas. However, there was no effect of sulfonylurea treatment after four weeks on 1.0-1.2 mg/kg/24 h glibenclamide., Conclusion: GAD, IA-2A, and ICA negative children with new onset type 1 diabetes have slower disease progression as assessed by residual beta-cell function and improved glycemic control 12 months after diagnosis. One out of 24 had a mutation in ABCC8, suggesting that screening of ABCC8 should be considered in patients with AAB negative type 1 diabetes.

Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.

Objective: Activating glucokinase (GCK) mutations are a rarely reported cause of congenital hyperinsulinism (CHI), but the prevalence of GCK mutations is not known., Methods: From a pooled cohort of 201 non-syndromic children with CHI from three European referral centres (Denmark, n=141; Norway, n=26; UK, n=34), 108 children had no K(ATP)-channel (ABCC8/KCNJ11) gene abnormalities and were screened for GCK mutations. Novel GCK mutations were kinetically characterised., Results: In five patients, four heterozygous GCK mutations (S64Y, T65I, W99R and A456V) were identified, out of which S64Y was novel. Two of the mutations arose de novo, three were dominantly inherited. All the five patients were medically responsive. In the combined Danish and Norwegian cohort, the prevalence of GCK-CHI was estimated to be 1.2% (2/167, 95% confidence interval (CI) 0-2.8%) of all the CHI patients. In the three centre combined cohort of 72 medically responsive children without K(ATP)-channel mutations, the prevalence estimate was 6.9% (5/72, 95% CI 1.1-12.8%). All activating GCK mutations mapped to the allosteric activator site. The novel S64Y mutation resulted in an increased affinity for the substrate glucose (S(0.5) 1.49+/-0.08 and 7.39+/-0.05 mmol/l in mutant and wild-type proteins respectively), extrapolating to a relative activity index of approximately 22 compared with the wild type., Conclusion: In the largest study performed to date on GCK in children with CHI, GCK mutations were found only in medically responsive children who were negative for ABCC8 and KCNJ11 mutations. The estimated prevalence (approximately 7%) suggests that screening for activating GCK mutations is warranted in those patients.

The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion in which the malignant cells resist digestion relative to nonmalignant cells. Treatment of T4-2 breast cancer cells in three-dimensional culture with cAMP analogs or a phosphatidylinositol 3-kinase inhibitor not only reverted their phenotype from nonpolar to polar acinar-like structures but also enhanced chromatin sensitivity to AluI. By using different cAMP analogs, we show that cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin produced the same effect. These experiments underscore the concept that modifying the tumor microenvironment can alter the organization of tumor cells and demonstrate that architecture and global chromatin organization are coupled and highly plastic.

The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment.

Protein phosphatase-directed toxins such as okadaic acid (OA) are general apoptosis inducers. We show that a protein (inhibitor of radiation- and OA-induced apoptosis, Irod/Ian5), belonging to the family of immune-associated nucleotide binding proteins, protected Jurkat T-cells against OA- and gamma-radiation-induced apoptosis. Unlike previously described antiapoptotic proteins Irod/Ian5 did not protect against anti-Fas, tumor necrosis factor-alpha, staurosporine, UV-light, or a number of chemotherapeutic drugs. Irod antagonized a calmodulin-dependent protein kinase II-dependent step upstream of activation of caspase 3. Irod has predicted GTP-binding, coiled-coil, and membrane binding domains. Irod localized to the centrosomal/Golgi/endoplasmic reticulum compartment. Deletion of either the C-terminal membrane binding domain or the N-terminal GTP-binding domain did not affect the antiapoptotic function of Irod, nor the centrosomal localization. The middle part of Irod, containing the coiled-coil domain, was therefore responsible for centrosomal anchoring and resistance toward death. Being widely expressed and able to protect also nonimmune cells, the function of Irod may not be limited to the immune system. The function and localization of Irod indicate that the centrosome and calmodulin-dependent protein kinase II may have important roles in apoptosis signaling.

A number of cyclin-dependent protein kinase (CDK) inhibitors were tested for the ability to protect IPC-81 rat leukemic cells against cAMP-induced apoptosis. A near perfect proportionality was observed between inhibitor potency to protect against cAMP-induced apoptosis and to antagonize CDK5, and to a lesser extent, CDK2 and CDK1. Enforced expression of dominant negative CDK5 (but not CDK1-dn or CDK2-dn) protected against death, indicating that CDK5 activity was necessary for cAMP-induced apoptosis. The CDK inhibitors failed to protect the cells against daunorubicine-, staurosporine-, or okadaic acid-induced apoptosis. The inhibition of CDK5 prevented the cleavage of pro-caspase-3 in cAMP-treated cells. The cells could be saved closer to the moment of their onset of death by inhibitors of caspases than by inhibitors of CDK5. This suggested that the action of CDK5 was upstream of caspase activation. The cAMP treatment resulted in a moderate increase of the level of CDK5 mRNA and protein in IPC-81 wild-type cells. Such cAMP induction of CDK5 was not observed in cells expressing the inducible cAMP early repressor. The cAMP-induced increase of CDK5 contributed to apoptosis since cells overexpressing CDK5-wt were more sensitive for cAMP-induced death. These results demonstrate the first example of a proapoptotic CDK action upstream of caspase activation and of an extra-neuronal effect of CDK5.

Recombinant beta-1,4-galactanase from Aspergillus aculeatus has been crystallized and characterized by X-ray diffraction. Crystals were obtained in hanging drops by vapour-diffusion under the conditions 30% PEG 400, 0.2 M CaCl2 and 0.1 M Na HEPES buffered to pH 7.5. The crystals diffract to 2.3 A resolution and belong to one of the orthorhombic space groups I222 or I212121. The unit-cell dimensions are a = 60.42, b = 88.94 and c = 129.08 A. With one molecule in the asymmetric unit, the corresponding solvent content is approximately 48%.

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4.5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 alpha and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters. Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

Penicillium freii (Lund and Frisvad 1994) mutants deficient in the synthesis of xanthomegnin were isolated. In vitro translated mRNA populations from selected radiation induced mutants and naturally occurring P. freii strains not able to produce xanthomegnin were examined by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Specific translation products were absent in mutants and natural isolates unable to produce xanthomegnin metabolites. One mutant (TSM 73) did not produce several of these translation products, indicating that a mutation in a regulatory gene involved in xanthomegnin production had occurred.

Expression cloning has been used to isolate a cDNA encoding beta-1,4-galactanase from the filamentous fungus Aspergillus aculeatus. A cDNA library was prepared from mycelia, inserted in a yeast expression vector and transformed into Saccharomyces cerevisiae. Thirteen clones secreting galactanase activity were identified from a screening of approximately 2.5 x 10(4) yeast colonies. All clones expressed transcripts of the same galactanase gene. The cDNA was re-cloned in an Aspergillus expression vector and transformed into Aspergillus oryzae. The recombinant enzyme had a molecular weight of 44,000 Da, an isoelectric point of pH 2.85, a pH optimum of pH 4.0-4.5, and a temperature optimum of 45-65 degrees C, which is similar to values obtained for a beta-1,4-galactanase purified from A. aculeatus. The enzyme degraded unsubstituted galactan to galactose and galactobiose. The deduced primary sequence of the enzyme showed no apparent homology to any known enzyme, in accordance with this being the first reported beta-1,4-galactanase cDNA. However, the deduced amino-acid sequence of a Bacillus circulans DNA sequence containing an open reading frame (ORF) with no known function, showed 36% identity and 60% similarity to the galactanase amino-acid sequence.