Cancer Metabolism: Fasting Reset, the Keto-Paradox and Drugs for Undoing.

In tumor cells, ketolysis "via" succinyl-CoA: 3-oxoacid-CoAtransferase (SCOT) and acetyl-CoA acetyltransferase 1 (ACAT1) is a major source of mitochondrial acetyl-CoA. Active ACAT1 tetramers stabilize by tyrosine phosphorylation, which facilitates the SCOT reaction and ketolysis. Tyrosine phosphorylation of pyruvate kinase PK M2 has the opposite effect, stabilizing inactive dimers, while pyruvate dehydrogenase (PDH), which is already inhibited by phosphorylation, is acetylated by ACAT1 and is doubly locked. This closes the glycolytic supply of acetyl-CoA. In addition, since tumor cells must synthesize fatty acids to create new membranes, they automatically turn off the degradation of fatty acids into acetyl-CoA ("via" the malonyl-CoA brake for the fatty acid carnityl transporter). Thus, inhibiting SCOT the specific ketolytic enzyme and ACAT1 should hold back tumor progression. However, tumor cells are still able to take up external acetate and convert it into acetyl-CoA in their cytosol "via" an acetyl-CoA synthetase, which feeds the lipogenic pathway; additionally, inhibiting this enzyme would make it difficult for tumor cells to form new lipid membrane and survive.

Labeling Antibodies.

This introduction outlines general strategies for labeling proteins, with an emphasis on methods that are used primarily for labeling antibodies. It covers the specific site of modification, cross-linker options, types of labels, and postlabeling cleanup methodology, along with the advantages and disadvantages of each method. In general, polyclonal antibodies are more versatile and resistant to activity loss than are monoclonal antibodies. Greater care must be taken when labeling monoclonal antibodies to ensure a quality conjugate. The methods outlined here can be adapted for a variety of labels including multiple labels on the same immunoglobulin. The most important consideration when undertaking an antibody labeling experiment is to maintain the activity of the antibody. This is an empirical process and will often require additional experiments to optimize the label of a particular antibody. When successful, these reagents are very useful and adaptable biomolecules. This introduction provides the reader with methods and options for producing a variety of labeled immunological tools.

An In Vitro Comparison of Estrogenic Equivalents Per Serving Size of Some Common Foods.

The public assumes that some foods, such as milk and ground beef from cattle receiving steroidal implants, are associated with estrogenic hormones, while other foods are presumed "safe" or nonestrogenic. Here, we investigate these assumptions by assessing the relative estrogenic activity of a serving size of four foods: skim milk (8 oz), rice (48 g dry wt) in cooking bag, ground beef patties from steers raised with or without hormone implantation (quarter lb each, 114 g), and tofu burgers (isocaloric to beef burger, 198 g), using an in vitro assay (E-Screen). Mean picogram (pg) estradiol equivalents (E2Eqs) on a serving basis were as follows: skim milk 120; rice 400; rice prepared in cooking bag 370; rice boiling bag alone 4 pg per bag, ground beef burger (obtained from the tissue of cattle that had received no hormone implants) 389, beef burger (obtained from cattle that had received hormone implant) 384, and tofu burger 1,020,000. Rice E2Eqs were highly variable, but the plastic cooking bags provided by the manufacturer added negligible E2Eqs. The source of estrogenic activity in rice may have been due to contamination with the mycotoxin zearalenone. The E-Screen E2Eqs of tofu burger extracts agreed with those predicted based on chemical concentrations of the most estrogenic component times their E2Eq factor. While a tofu burger contained around three times the estrogenic activity of a daily dose of estrogen replacement therapy (125 mg, Premarin®, 303,000 pg); the other foods--a quarter pound ground beef burger at approximately equal calorie count, a serving of milk, or rice, were all at least 750-fold less estrogenic. PRACTICAL APPLICATION: When consuming the recognized serving size of a food, how much estrogenic activity can we expect? While the public assumes that some foods, such as milk and ground beef from cattle receiving steroidal implants, are associated with estrogenic hormones, other foods are presumed "safe" or nonestrogenic. Using one assay, a tofu burger contained three times the estrogenic activity of a dose of hormone replacement therapy commonly prescribed for women after hysterectomy or menopause (Premarin®); while other foods--a quarter pound ground beef burger at approximately equal calorie count, a serving of milk, or rice, were all at least 750-fold less estrogenic.

Conjugation of Peptides to Thiol-Reactive Gel for Affinity Purification of Antibodies.

Thiol-reactive linkers, such as iodoacetyl or maleimide, bound to cross-linked agarose are used to attach cysteine-containing peptides covalently to this resin for use in affinity-purification protocols. It is critical to confirm that the peptide contains a reduced cysteine so that the thiol is available for conjugation to the thiol-reactive linker. The column should be sized appropriately for the amount of peptide to be used and the volume of serum to be processed. Excess binding sites on the column must be blocked with free cysteine before use.

Peptide Affinity Purification of Antibodies.

This protocol describes antibody purification using a peptide affinity column. Peptides can be designed that use naturally occurring cysteines within the protein target's primary sequence, or a cysteine can be added to either end of the peptide to provide free thiols for attachment. The peptides can then be covalently attached to resins bearing thiol-reactive linkers. The most commonly used thiol-reactive moieties are iodoacetyl and maleimide, both of which react selectively with peptides containing cysteine thiols. Although gravity can be used to cycle the antibody solution (e.g., serum) over the column (it is recommended that the antibody be cycled multiple times to obtain maximal yield), the use of a pump to apply the serum to the column in a continuous flow manner improves the yield of antibody. Similarly, washing the column after application of the antibody without and with 0.5 m NaCl should be performed with at least 20 column volumes.

Purification of Antibodies: Diethylaminoethyl (DEAE) Chromatography.

Because IgG from most species (other than rodents) tends to have an isoelectric point around neutral, two approaches can be used when separating IgG using diethylaminoethyl (DEAE) resins. When serum containing antibodies is applied to DEAE at a slightly acidic pH, the IgG flows through the column while most other serum proteins bind to the DEAE. This method is best performed using a batch method. The DEAE beads can be kept in a disposable syringe containing a polypropylene frit, a glass reactor containing a coarse-sintered glass frit, or other suitable vessel. If the antibody solution is adjusted to a basic pH of 8-8.5, then IgG binds to the DEAE resin. After washing the column, the antibody is eluted by adding a buffer of increasing ionic strength to the column. Prepacked columns of many sizes are available for the isolation of antibodies by DEAE chromatography. Alternatively, DEAE medium can be swelled or prepped according to the manufacturer's instructions and a column can be poured when needed.

Intracranial complications of acute sinusitis in children: The role of endoscopic sinus surgery.

OBJECTIVE: To study the role of endoscopic sinus surgery (ESS) in the management of intracranial complications of children with acute rhinosinusitis METHODS: Retrospective chart review at a tertiary care pediatric hospital MAIN OUTCOMES: Demographics, intracranial complications, length of hospital stay (LOS), neurological sequelae, ESS, neurosurgical procedures RESULTS: Twenty-four children with a mean age (SD) of 12.9 years (+/-3.2) with an intracranial complication(s) of acute rhinosinusitis were identified between 2005-2016. A total of 22 were included and 15 (68%) of these were males. The most common complications were: subdural abscess (n=10), epidural abscess (n=10), meningitis (n=5), intraparenchymal abscess (n=5), and cavernous sinus thrombosis (n= 2). Neurologic symptoms included headache (n=12), hemiparesis (n=5) and aphasia (n=3). Average length of stay was 16 (+/- 9.2) days. Average follow up was 7 (+/-5.6) months. One patient had residual seizures and 1 had recurrent rhinosinusitis. Aphasia and hemiparesis resolved in all patients within 1 year. Nineteen (86%) patients had ESS within 4 days of admission. Fourteen patients (63%) had a neurosurgical procedure, 6 (27%) required more than 1 neurosurgical procedure. Six patients (27%) had concurrent neurosurgical drainage and ESS. Four patients (17%) had neurosurgical procedure followed by ESS and 3 patients (13%) were treated only by a neurosurgical procedure. Patients who underwent ESS prior to a neurosurgical procedure had significantly less risk of needing a neurosurgical intervention (OR = .02, p < .01). There was a significantly higher proportion of neurosurgical patients with positive Strep anginosus cultures compared to the ESS only group (85.7% vs 37.5%, p = .02). Studies with larger patient populations are needed to determine the role of ESS in the management of intracranial complications of children with acute rhinosinusitis. DISCUSSION: Early ESS may be associated with less need for neurosurgical procedures.

Maxillary sinus mucoceles and other side effects of external-beam radiation in the pediatric patient: A cautionary tale.

A sinonasal mucocele can develop after the obstruction of a sinus ostium, which can occur secondary to trauma, infection, a neoplasm, or an iatrogenic cause. These mucoceles typically arise in the frontal and ethmoid sinuses. Sinonasal mucoceles are epithelium-lined cysts that can gradually expand to fill a paranasal sinus. They can present with ophthalmologic, rhinologic, and neurologic symptoms. There are reports in the literature of sinonasal mucocele development after radiation for nasopharyngeal carcinoma. However, these cases are rare; when they do occur, they tend to arise in the sphenoid sinus. We describe the case of a 21-year-old man who had undergone external-beam radiotherapy for a right maxillary sinus rhabdomyosarcoma at the age of 4 years and who subsequently developed bilateral maxillary sinus mucoceles and multiple other known complications of radiation therapy. To the best of our knowledge, this is the first case of postirradiation bilateral maxillary sinus mucoceles to be reported in the literature.

Isolated epiglottic lymphatic malformation in a 2-year-old: Diagnosis and treatment.

Airway obstruction in children has a wide differential diagnosis that includes laryngomalacia, infectious processes, paralysis, extrinsic compression, and other rare anatomic anomalies. Isolated laryngeal lymphatic malformations are rare developments that can manifest with clinically significant airway obstruction. To the best of our knowledge, there have been fewer than 20 reported cases. These laryngeal mucosal lesions are best managed with radiofrequency ablation or laser ablation. We present a case of a 2-year-old child who presented with airway obstruction, initially diagnosed with laryngomalacia, who was subsequently diagnosed and treated for an isolated epiglottic lymphatic malformation.

Consumption of ground beef obtained from cattle that had received steroidal growth promotants does not trigger early onset of estrus in prepubertal pigs.

BACKGROUND: The earlier onset of puberty seen in young American girls has led researchers to question if a causal relation exists between dietary sources of estrogenic compounds and precocious puberty. OBJECTIVE: Using the prepubertal gilt (young female pig) as an animal model, our hypothesis is that feeding beef obtained from cattle receiving growth-promoting steroidal implants postweaning does not alter the onset of puberty or the peripubertal body composition of gilts compared with contemporaries fed nonimplanted "natural" beef or a common meat alternative, tofu. METHOD: The base diet was formulated using canola meal replacing soybean meal to reduce diet estrogenicity. Feed intake was monitored and controlled to ensure similar intake. Gilts were assigned to treatments based on dam and initial body weight (mean: 24.5 ± 3.20 kg) at 61 d of age. The negative control base diet was supplemented with daily feedings of a cooked patty from nonimplanted steers (natural), from steers that had been treated with growth promotants [100 mg trenbolone acetate and 14 mg estradiol (E2) benzoate; implanted], or cooked tofu patty. RESULTS: E2 equivalents (nanogram per kilogram, as fed as analyzed by E-Screen) of the tofu (a soy-based product) supplement were ∼570 times the natural and ∼170 times the implanted supplements. There were no observed differences across treatments in live weight gain (P = 0.90), longissimus muscle area developed at the 10th and 11th rib interface (P = 0.46), and subcutaneous fat deposition (P = 0.41) at the same location over time or in the number of days to reach estrus (P = 0.55). CONCLUSIONS: Consumption of beef from growth implanted or natural steers or tofu at levels similar to those typically consumed by humans did not impact growth or onset of estrus in these prepubertal gilts.

Effect of cassava bioethanol by-product and crude palm oil in Brahman x Thai native yearling heifer cattle diets: I. Nutrient digestibility and growth performance.

The effects of cassava bioethanol by-product (CEP) and crude palm oil (CPO) on feed intake, nutrient digestibility, and growth performance of yearling heifers were investigated in a 150-day feeding trial. Eighteen, crossbred heifers (Brahman x Thai native) were randomly allotted according to 2 × 3 factorial arrangement. Low or high levels of CEP (15 or 30% of concentrate, LCEP, or HCEP) were basal treatments and 0, 2, and 4% CPO were daily top-dressed. Concentrate was supplemented at 1.75% of body weight (BW) and rice straw offered ad libitum. CEP level had no significance on feed intake. CPO increased roughage intake, concentrate intake, and total feed intake when expressed as %BW/d (P < 0.01) and as metabolic BW (kg(0.75)/d, P < 0.05). Intakes of dry matter (DM), organic matter (OM), and crude protein (CP) were similar (P > 0.05). Intake of fat increased with higher levels of CPO (P < 0.001). The DM, OM, CP, and EE digestibility of cattle-fed HCEP was lower than LCEP, but adding 4% CPO increased digestibility. Growth performance was similar for all diets (P > 0.05). We concluded that CEP can be used up to 30% in the diet, with or without additional fat inclusion.

Role of CRD-BP in the growth of human basal cell carcinoma cells.

Although the number of new cases of basal cell carcinoma (BCC) has increased rapidly in the last few decades, the molecular basis of its pathogenesis is not completely understood. Activation of the Hedgehog (Hh) signaling pathway has been shown to be a key factor driving the development of BCC. The Wnt/ß-catenin signaling pathway was also shown to be activated in BCCs and to perhaps modulate the activity of the Hh pathway. We have previously identified a mechanism by which Wnt signaling regulates the transcriptional outcome of the Hh signaling pathway. We demonstrated that coding region determinant-binding protein (CRD-BP), a direct target of the Wnt/ß-catenin signaling, binds to GLI1 mRNA, stabilizes it, and consequently upregulates its levels (mRNA and protein) and activities. We hypothesized that Wnt-induced and CRD-BP-dependent regulation of GLI1 expression and activities is important for the development of BCC. In this study, we show that CRD-BP is overexpressed in BCC and that its expression positively correlates with the activation of both Wnt and Hh signaling pathways. We also describe the generation and characterization of a human BCC cell line. This cell line was utilized to demonstrate the importance of CRD-BP-dependent regulation of GLI1 expression and activities in the development of BCC.

Endoscopic cauterization of congenital pyriform fossa sinus tracts: an 18-year experience.

IMPORTANCE: Congenital pyriform fossa sinus tracts predispose to neck masses and neck abscesses in pediatric and occasionally adult patients. Traditional management involves open excision with substantial potential morbidity. Endoscopic management allows an alternative, less morbid treatment approach. OBJECTIVE: To evaluate the long-term effectiveness of endoscopic cauterization as definitive treatment for pyriform fossa sinus tracts. DESIGN, SETTING, AND PATIENTS: Retrospective review of the medical records of 23 children (aged 7 months to 14 years) with pyriform fossa sinus tracts treated with endoscopic cauterization between 1995 and 2013 at a tertiary care children's hospital. INTERVENTION: Endoscopic electrocauterization of pyriform fossa sinus tract opening. MAIN OUTCOMES AND MEASURES: Recurrence of symptoms after endoscopic treatment. RESULTS: Twenty-one of 23 patients experienced no recurrence after their first endoscopic electrocauterization of the sinus tract. The 2 patients with recurrence experienced symptoms within 1 month of cauterization and were treated with either open excision or recauterization. Endoscopic cauterization was able to definitively treat 9 patients whose treatments with incision and drainage or open excision had failed. Mean (range) follow-up for the 15 patients with follow-up was 7.4 (0.10-14.2) years. No procedure-related morbidity was reported. CONCLUSIONS AND RELEVANCE: Endoscopic cauterization seems to be an effective and potentially permanent treatment for congenital pyriform fossa sinus tracts.

Laryngeal obstruction by cervical and endolaryngeal lymphatic malformations in children: proposed staging system and review of treatment.

OBJECTIVES: We review and stage a series of congenital lymphatic malformations (LMs) that caused laryngeal obstruction according to a proposed staging system, review the treatment modalities utilized, and correlate the stage of disease with clinical outcomes and tracheotomy statuses. METHODS: We present a detailed description of the clinical presentation, management approach, and clinical outcome and tracheotomy status of a series of patients with laryngeal obstruction by LMs. A 4-part staging system (stages I to IV) for congenital LMs obstructing the larynx, based upon our clinical experience, is proposed. The patients were retrospectively reviewed and staged according to the proposed system, and clinical management and outcomes were correlated with the stage of disease. RESULTS: We identified 16 patients with laryngeal obstruction by LMs. Eighty-one percent (13 of 16) received sclerotherapy, and 50% (8 of 16) underwent operative excision or debridement. Forty percent (2 of 5) of stage I lesions, 75% (3 of 4) of stage II lesions, 100% (4 of 4) of stage III lesions, and 100% (3 of 3) of stage IV lesions were in patients who required tracheotomy. All patients who had stage I and II lesions and required tracheotomy have been decannulated, whereas only 2 of the 4 patients with stage III lesions and no patients with stage IV lesions have been successfully decannulated. CONCLUSIONS: Lymphatic malformations obstructing the larynx require a careful and often staged management approach. A proposed staging system helps to predict the need for tracheotomy and the likelihood of long-term tracheotomy dependence.

Radiation fibrosis of the vocal fold: from man to mouse.

OBJECTIVES/HYPOTHESIS: To characterize fundamental late tissue effects in the human vocal fold following radiation therapy. To develop a murine model of radiation fibrosis in order to ultimately develop both treatment and prevention paradigms. DESIGN: Translational study using archived human and fresh murine irradiated vocal fold tissue. METHODS: 1) Irradiated vocal fold tissue from patients undergoing laryngectomy for loss of function from radiation fibrosis was identified from pathology archives. Histomorphometry, immunohistochemistry, and whole-genome microarray, as well as real-time transcriptional analyses, were performed. 2) Focused radiation to the head and neck was delivered to mice in a survival fashion. One month following radiation, vocal fold tissue was analyzed with histomorphometry, immunohistochemistry, and real-time PCR transcriptional analysis for selected markers of fibrosis. RESULTS: Human irradiated vocal folds demonstrated increased collagen transcription, with increased deposition and disorganization of collagen in both the thyroarytenoid muscle and the superficial lamina propria. Fibronectin were increased in the superficial lamina propria. Laminin decreased in the thyroarytenoid muscle. Whole genome microarray analysis demonstrated increased transcription of markers for fibrosis, oxidative stress, inflammation, glycosaminoglycan production, and apoptosis. Irradiated murine vocal folds demonstrated increases in collagen and fibronectin transcription and deposition in the lamina propria. Transforming growth factor (TGF)-ß increased in the lamina propria. CONCLUSION: Human irradiated vocal folds demonstrate molecular changes leading to fibrosis that underlie loss of vocal fold pliability occurring in patients following laryngeal irradiation. The irradiated murine tissue demonstrates similar findings, and this mouse model may have utility in creating prevention and treatment strategies for vocal fold radiation fibrosis.

Peptide-conjugated PAMAM dendrimer as a universal DNA vaccine platform to target antigen-presenting cells.

DNA-based vaccines hold promise to outperform conventional antigen-based vaccines by virtue of many unique features. However, DNA vaccines have thus far fallen short of expectations, due in part to poor targeting of professional antigen-presenting cells (APC) and low immunogenicity. In this study, we describe a new platform for effective and selective delivery of DNA to APCs in vivo that offers intrinsic immune-enhancing characteristics. This platform is based on conjugation of fifth generation polyamidoamine (G5-PAMAM) dendrimers, a DNA-loading surface, with MHC class II-targeting peptides that can selectively deliver these dendrimers to APCs under conditions that enhance their immune stimulatory potency. DNA conjugated with this platform efficiently transfected murine and human APCs in vitro. Subcutaneous administration of DNA-peptide-dendrimer complexes in vivo preferentially transfected dendritic cells (DC) in the draining lymph nodes, promoted generation of high affinity T cells, and elicited rejection of established tumors. Taken together, our findings show how PAMAM dendrimer complexes can be used for high transfection efficiency and effective targeting of APCs in vivo, conferring properties essential to generate effective DNA vaccines.

Pathologic effects of external-beam irradiation on human vocal folds.

OBJECTIVES: We sought to better characterize pathologic changes that occur in the human vocal fold after radiotherapy for head and neck cancer. METHODS: In a blinded, controlled study of archived tissue, we evaluated postirradiation salvage laryngectomy vocal fold tissue without evidence of malignant disease. Clinical and demographic patient data were collected. In a blinded fashion, irradiated tissue was compared to nonirradiated, benign control tissue. Histomorphometric analysis was used to assess muscle and collagen organization, superficial lamina propria (SLP) and vocal ligament thickness, vocalis muscle fiber area, collagen content, and hyaluronic acid content. Immunohistochemical analysis was used to assess the content of type I collagen, type IV collagen, vimentin, fibronectin, alpha-smooth muscle actin, matrix metalloproteinase 9, and laminin. RESULTS: Twenty irradiated vocal folds were evaluated and compared to control specimens. Collagen and muscle disorganization was noted in the irradiated specimens. The SLP and vocal ligament thicknesses and the mean muscle fiber diameters did not differ significantly. The SLP fibronectin and the vocalis muscle and SLP collagen content were significantly increased in the irradiated vocal folds, and the SLP collagen content increased significantly with time between irradiation and resection. The laminin content of irradiated vocalis muscles was significantly decreased. CONCLUSIONS: Radiotherapy results in significant vocal fold tissue changes. Having more precisely defined these changes, we plan continued investigation seeking targeted preventive and therapeutic interventions for improved vocal quality following radiotherapy.