Age-related decline in goblet cell numbers and mucin content of the human colon: Implications for lower bowel functions in the elderly.

BACKGROUND & AIMS: Older people experience a greater incidence of lower bowel disorders, including constipation. Causes can include factors associated with growing older, such as use of medications or disease, but compounded by degenerative changes within the bowel wall. It has been suggested that the latter is exacerbated by loss of an effective mucosal barrier to luminal contents. In human colon, little is known about the impact of ageing on key components of this barrier, namely the goblet cells and mucin content. METHODS: Changes in the number of goblet cells and density of mucin content were investigated in macroscopically normal human ascending (AC; n = 13) and descending (DC; n = 14) colon from elderly (≥ 67 years) and younger adults (60 years and below). Samples were serially sectioned and stained for haematoxylin and eosin to assess tissue morphology, and alcian blue periodic acid Schiff (ABPAS) and MUC-2 antibody to identify goblet cells producing mucins. New procedures in visualization and identification of goblet cells and mucin contents were employed to ensure unbiased counting and densitometric analysis. RESULTS: Compared with the younger adults, the numbers of goblet cells per crypt were significantly lower in the elderly AC (72 ± 1.2 vs 51 ± 0.5) and DC (75 ± 2.6 vs. 54 ± 1.9), although this reduction did not reach statistical significance when assessed per mucosal area (AC: P = 0.068; DC: P = 0.096). In both regions from the elderly, numerous empty vesicles (normally containing mucins) were observed, and some areas of epithelium were devoid of goblet cells. Thus, the density of mucin content per unit mucosal area were significantly reduced with age. CONCLUSIONS: Ageing could result in a reduced number of goblet cells and development of degenerative changes in mucin production. Together, these have implications for the mucus barrier function in the colon of elderly individuals.

A rare cutaneous infection with Mycobacterium chelonae in a pediatric patient treated with trametinib for KRAS-mutated diffuse glioma.

Mitogen-activated extracellular signal-regulated kinase inhibitors (MEKi) represent a promising new therapy for pediatric patients with low-grade gliomas, which frequently have abnormal signaling within the mitogen-activated protein kinase (MAP kinase) pathway. However, understanding of long-term efficacy and toxicity is limited in pediatric glioma patients. This article describes a rare presentation of a widespread cutaneous infection with Mycobacterium chelonae in a pediatric patient with a low-grade glioma treated with trametinib.

Appropriate use criteria for ancillary diagnostic testing in dermatopathology: New recommendations for 11 tests and 220 clinical scenarios from the American Society of Dermatopathology Appropriate Use Criteria Committee.

BACKGROUND: Appropriate use criteria (AUC) provide patient-centered physician guidance in test selection. An initial set of AUC was reported by the American Society of Dermatopathology (ASDP) in 2018. AUC reflect evidence collected at single timepoints and may be affected by evolving evidence and experience. The objective of this study was to update and expand AUC for selected tests. METHODS: RAND/UCLA (RAND Corporation [Santa Monica, CA]/University of California Los Angeles) methodology used includes the following: (a) literature review; (b) review of previously rated tests and previously employed clinical scenarios; (c) selection of previously rated tests for new ratings; (d) development of new clinical scenarios; (e) selection of additional tests; (f) three rating rounds with feedback and group discussion after rounds 1 and 2. RESULTS: For 220 clinical scenarios comprising lymphoproliferative (light chain clonality), melanocytic (comparative genomic hybridization, fluorescence in situ hybridization, reverse transcription polymerase chain reaction, telomerase reverse transcriptase promoter), vascular disorders (MYC), and inflammatory dermatoses (periodic acid-Schiff, Gömöri methenamine silver), consensus by panel raters was reached in 172 of 220 (78%) scenarios, with 103 of 148 (70%) rated "usually appropriate" or "rarely appropriate" and 45 of 148 (30%), "appropriateness uncertain." LIMITATIONS: The study design only measures appropriateness. Cost, availability, test comparison, and additional clinical considerations are not measured. The possibility that the findings of this study may be influenced by the inherent biases of the dermatopathologists involved in the study cannot be excluded. CONCLUSIONS: AUC are reported for selected diagnostic tests in clinical scenarios that occur in dermatopathology practice. Adhering to AUC may reduce inappropriate test utilization and improve healthcare delivery.

Brain Lesions in Aging Zoo-Housed Naked Mole-Rats (Heterocephalus glaber).

Naked mole-rats (NMRs) are common in the managed care of zoos and valuable models for aging research. Limited information on NMR neuropathology is available despite many studies regarding their aging physiology. Histologic sections of brain from 27 adult (5-27 years old) NMRs from 2 zoos were reviewed to determine presence or absence of lesions associated with advanced age in humans and other mammals. A majority (23/27; 85%) of NMR brains had cerebral cortical neuronal changes with rounded or angular neurons, cytoplasmic vacuoles containing pale yellow pigment, periodic acid-Schiff (PAS)-positive granules and green autofluorescence, compatible with lipofuscinosis. Less severe lesions were present in cerebellar Purkinje cells, medulla, and hippocampal neurons. The hypothalamic neuropil of all NMRs had scattered variably sized PAS-positive granules and 10 (37%) had larger round bodies consistent with corpora amylacea. The youngest NMRs, 5 to 7 years old, generally had minimal or no cerebrocortical lesions. Further studies will help understand brain aging in this long-lived species.

Diagnostic value of fluorescein-labeled chitinase staining in formalin-fixed and paraffin-embedded tissues of fungal disease.

Common histopathologic techniques are used to diagnose fungal infections, but the diagnostic identification of mycoses in tissue specimens is often difficult, particularly when fungi rarely occur in a specimen. The aim of this study was to evaluate the application of fluorescein-labeled chitinase staining to formalin-fixed and paraffin-embedded (FFPE) tissues. We studied 79 archival FFPE tissues from patients diagnosed with fungal disease, including 38 cases of sporotrichosis and 41 cases of other fungal infections. The tissue sections were subjected to periodic acid-Schiff (PAS) staining, Gomori's methenamine silver (GMS) staining, and fluorescein-labeled chitinase staining to detect fungal elements. Culture- and/or hematoxylin-eosin-positive samples were used to estimate the diagnostic sensitivity of each staining method, with the results showing that PAS, GMS, and fluorescein-labeled chitinase staining had sensitivities of 50.6, 70.9, and 68.4%, respectively. The three staining results were the same for all fungal infections except for sporotrichosis and chromoblastomycosis. Fluorescein-labeled chitinase staining exhibited high sensitivity in cases of sporotrichosis and poor performance in detecting muriform cells of chromoblastomycosis. On the whole, the sensitivity of fluorescein-labeled chitinase staining was greater than that of PAS and similar to that of GMS staining. Therefore, the results of our study suggest that fluorescein-labeled chitinase staining is a potentially useful diagnostic tool in the diagnosis of fungal infections.

Whipple disease diagnosed by enteroscopy: first case report in Colombia of an underdiagnosed disease and literature review.

BACKGROUND: Whipple's disease is a rare systemic disease caused by a gram-positive bacillus called Tropheryma whipplei. First described in 1907 as an intestinal lipodystrophy with histological finding of vacuoles in the macrophages of the intestinal mucous. Usually the symptoms are localized according to the compromised organ. The differential diagnosis is wide. It can be fatal without proper treatment. Recurrence can occur in up to 33% of the cases and usually compromises the neurological system. CASE PRESENTATION: This article reports the case of a 46-year-old female patient with a history of a 6-month hypochromic microcytic anemia of unknown cause. She consulted for a 6-months oppressive abdominal pain located in the mesogastrium as well as abdominal distention associated with nausea and liquid stools; in addition, she had an 8-month small and medium joint pain, without edema or erythema. Physical examination without relevant findings. Multiple esophagogastroduodenoscopies with normal gastric and duodenal biopsies findings and a normal colonoscopy were performed. Endoscope capsule showed red spots in the duodenum and ulcerations in the jejunum and proximal ileum covered by fibrin; histological report showed macrophages with positive periodic acid-schiff reaction staining (PAS staining), disgnosing Whipple's disease. Antibiotics were initiated. The patient is currently in the second phase of treatment without gastrointestinal and joint symptoms. CONCLUSION: This is the first case reported in Colombia. It is a rare entity and difficult to diagnose reason why it is important to continue with clinical investigations to give more clarity about the onset and appropriate diagnose to avoid the delay in treatment of this entity.

Pulmonary cryptococcosis coexisting with central type lung cancer in an immuocompetent patient: a case report and literature review.

BACKGROUND: Pulmonary Cryptococcosis is a common fungal infection mainly caused by Cryptococcus neoformans/C.gattii species in immunocompromised patients. Cases of pulmonary cryptococcosis in patients with normal immune function are increasingly common in China. Clinical and radiographic features of pulmonary cryptococcosis are various and without obvious characteristics, so it is often misdiagnosed as pulmonary metastatic tumor or tuberculosis. When coexisting with malignant lung tumors, it was more difficult to differentiate from metastatic lung cancer, although the coexistence of pulmonary cryptococcosis and central type lung cancer is rare. Reviewing the imaging manifestations and diagnosis of the case and the relevant literature will contribute to recognition of the disease and a decrease in misdiagnoses. CASE PRESENTATION: A 72-year-old immunocompetent Han Chinese man had repeated dry cough for more than half a year. CT examination of chest showed an irregular mass at the left hilum of the lung, and two small nodules in the right lung, which were considered as the left central lung cancer with right lung metastasis. However, the patient was diagnosed with pulmonary cryptococcosis coexisting with central type lung cancer based on the results of laboratory examination, percutaneous lung biopsy, fiberoptic bronchoscopy, and surgical pathology. The patient underwent surgical resection of the left central type lung cancer and was placed on fluconazole treatment after a positive diagnosis was made. Five years after the lung cancer surgery, the patient had a recurrence, but the pulmonary cryptococcus nodule disappeared. CONCLUSION: Our case shows that CT findings of central type lung cancer with multiple pulmonary nodules are not necessarily metastases, but may be coexisting pulmonary cryptococcosis. CT images of cryptococcosis of the lung were diverse and have no obvious characteristics, so it was very difficult to distinguish from metastatic tumors. CT-guided percutaneous lung biopsy was a simple and efficient method for identification.

Machine learning-based prediction of outcomes of the endoscopic endonasal approach in Cushing disease: is the future coming?

OBJECTIVE: Machine learning (ML) is an innovative method to analyze large and complex data sets. The aim of this study was to evaluate the use of ML to identify predictors of early postsurgical and long-term outcomes in patients treated for Cushing disease (CD). METHODS: All consecutive patients in our center who underwent surgery for CD through the endoscopic endonasal approach were retrospectively reviewed. Study endpoints were gross-tumor removal (GTR), postsurgical remission, and long-term control of disease. Several demographic, radiological, and histological factors were assessed as potential predictors. For ML-based modeling, data were randomly divided into 2 sets with an 80% to 20% ratio for bootstrapped training and testing, respectively. Several algorithms were tested and tuned for the area under the curve (AUC). RESULTS: The study included 151 patients. GTR was achieved in 137 patients (91%), and postsurgical hypersecretion remission was achieved in 133 patients (88%). At last follow-up, 116 patients (77%) were still in remission after surgery and in 21 patients (14%), CD was controlled with complementary treatment (overall, of 131 cases, 87% were under control at follow-up). At internal validation, the endpoints were predicted with AUCs of 0.81-1.00, accuracy of 81%-100%, and Brier scores of 0.035-0.151. Tumor size and invasiveness and histological confirmation of adrenocorticotropic hormone (ACTH)-secreting cells were the main predictors for the 3 endpoints of interest. CONCLUSIONS: ML algorithms were used to train and internally validate robust models for all the endpoints, giving accurate outcome predictions in CD cases. This analytical method seems promising for potentially improving future patient care and counseling; however, careful clinical interpretation of the results remains necessary before any clinical adoption of ML. Moreover, further studies and increased sample sizes are definitely required before the widespread adoption of ML to the study of CD.

Effects of various fixatives and temperature on the quality of glycogen demonstration in the brain and liver tissues.

The visualization of glycogen deposits in cells and tissues is important for studying glycogen metabolism as well as diagnosis of glycogen storage diseases. Evidence suggests that the demonstration of glycogen can better be enhanced by factors such the choice of fixative and temperature during fixation. Here, we assessed efficacy of neutral buffered formalin (NBF), alcoholic formalin (AF) and paraformaldehyde (PFA) at 4 °C, 37 °C and 40 °C using Periodic Acid Schiff's staining method. Each liver specimen was fixed in NBF and AF while the brain tissues were fixed in NBF, AF and PFA. We found that there was a better PAS staining intensity with the liver tissues fixed in AF compared with NBF. Also, there was no difference in the quality of the staining for tissues fixed in AF at 37 °C, 4 °C and 40 °C, but fixation with NBF at 4 °C gave the best staining quality when compared with 40 °C and 37 °C. Furthermore, hippocampal tissues fixed in AF showed better quality of PAS staining compared with NBF and PFA. A significant increase in staining intensity was observed for PFA when compared with NBF. Superior staining intensity for PAS was observed at 4 °C for hippocampal tissues fixed with NBF, AF and PFA. Taken together our results show that AF at a temperature of 4 °C gave the best result. Hence, glycogen demonstration can better be enhanced by the choice of fixative and temperature during fixation.

Eupatilin downregulates phorbol 12-myristate 13-acetate-induced MUC5AC expression via inhibition of p38/ERK/JNK MAPKs signal pathway in human airway epithelial cells.

Chronic inflammatory airway diseases, such as chronic rhinosinusitis, chronic obstructive pulmonary disease, and asthma, are associated with excessive mucus production. Hence, the regulation of mucus production is important for the treatment of upper and lower airway diseases. Eupatilin is a pharmacologically active ingredient obtained from Artemisia asiatica Nakai (Asteraceae) and exerts potent anti-inflammatory, anti-allergic, and anti-tumor activities. In the present study, we investigated the effect of eupatilin on phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC and MUC5B expression in human airway epithelial cells. We found that eupatilin treatment significantly inhibited PMA-induced mucus secretion in PAS staining. In addition, qRT-PCR results showed that eupatilin dose-dependently decreased the mRNA expression of MUC5AC in human airway epithelial cells. Western blot and immunofluorescence assay also showed that PMA-induced protein expression of MUC5AC was inhibited by eupatilin treatment. Finally, we investigated MAPKs activity after stimulation with PMA using western blot analysis in human airway epithelial cells. The results showed that eupatilin downregulated the levels of phosphorylated p38, ERK, and JNK. In summary, the anti-inflammatory activities of eupatilin, characterized as the suppression of MUC5AC expression and secretion in human airway epithelial cells, were found to be associated with the inhibition of p38/ERK/JNK MAPKs signaling pathway of MUC5AC secretion.

Evaluation of Better Staining Method among Hematoxylin and Eosin, Giemsa and Periodic Acid Schiff-Alcian Blue for the Detection of Helicobacter pylori in Gastric Biopsies.

BACKGROUND: This study was undertaken to evaluate the preferred method (Giemsa or periodic acid Schiff-Alcian blue [PAS-AB] stains) of detecting Helicobacter pylori (H. pylori) in gastric mucosal biopsies in terms of sensitivity, specificity and applicability. To the best of my knowledge, this is the first report comparing Giemsa and PAS-AB staining for the detection of H. pylori in such biopsies. METHODS: The formalin-fixed paraffin-embedded blocks of 49 gastric biopsies from different patients were collected from the archive of anatomical pathology at King Abdulaziz Medical City, National Guard, Riyadh, Saudi Arabia. From each block, three slides were prepared and analysed using the hematoxylin and eosin (H&E), Giemsa and PAS-AB stains to detect the presence/absence of H. pylori, and the results were compared in terms of sensitivity, specificity and applicability. RESULTS: The majority of the biopsies in this study showed antrum-type gastric mucosa. Only 15 biopsies showed active gastritis, whereas the rest showed chronic gastritis. Three biopsies showed intestinal metaplasia. All were detected by PAS-AB stain, but only two-thirds were detected by H&E stain. Fifteen gastric biopsies showed H. pylori infection in general and in 13 of them, active gastritis cases were discovered. Fourteen out of these 15 H. pylori infection cases were detected by Giemsa stain, whereas only 13 cases were detected by H&E stain. PAS-AB stain showed the worst results since it demonstrated only 40% sensitivity and 67.65% specificity in H. pylori detection. CONCLUSION: Giemsa stain has better sensitivity and specificity in gastric H. pylori infection detection than PAS-AB. Therefore, using PAS-AB stain to detect H. pylori infection is not recommended.

Application of fungal fluorescent staining in oral candidiasis: diagnostic analysis of 228 specimens.

BACKGROUND: Several conventional methods, including fungal culture and periodic acid-Schiff (PAS) reagent staining, have been used to diagnose oral candidiasis. The aim of this study was to evaluate the efficacy of a novel method, fungal fluorescent staining, in relation to conventional protocols in the diagnosis of oral candidiasis. METHODS: We collected 106 oral swabs and 122 oral biopsy tissues from patients highly suspected with oral candidiasis. We applied fungal culture and periodic acid-Schiff reagent staining as the gold standard diagnostic tools. The efficacy of these methods in determining the presence of Candida was compared with that of fluorescent staining. RESULTS: In the majority of specimens subjected to fluorescent staining, fungal organisms were distinguished by blue fluorescence surrounding their tubular or annular shapes. The sensitivity, specificity, Youden index, positive predictive value and negative predictive value of the fluorescent staining method were 82.7, 93.5, 76.7, 96.8 and 69.1% in oral swabs and 90.0, 92.9, 82.9, 96.0 and 82.9% in oral biopsy tissues, respectively. CONCLUSIONS: Fungal fluorescent staining represents a rapid method for detection of Candida, supporting its potential utility as an effective early diagnostic tool for oral candidiasis.

Variations in internal structure, composition and protein distribution between intra- and extra-articular knee ligaments and tendons.

Tendons and ligaments play key roles in the musculoskeletal system in both man and animals. Both tissues can undergo traumatic injury, age-related degeneration and chronic disease, causing discomfort, pain and increased susceptibility to wider degenerative joint disease. To date, tendon and ligament ultrastructural biology is relatively under-studied in healthy, non-diseased tissues. This information is essential to understand the pathology of these tissues with regard to function-related injury and to assist with the future development of tissue-engineered tendon and ligament structures. This study investigated the morphological, compositional and extracellular matrix protein distribution differences between tendons and ligaments around the non-diseased canine stifle joint. The morphological, structural characteristics of different regions of the periarticular tendons and ligaments (the intra-articular anterior cruciate ligament, the extra-articular medial collateral ligament, the positional long digital extensor tendon and energy-storing superficial digital flexor tendons) were identified using a novel semi-objective histological scoring analysis and by determining their biochemical composition. Protein distribution of extracellular matrix collagens, proteoglycans and elastic fibre proteins in anterior cruciate ligament and long digital extensor tendon were also determined using immunostaining techniques. The anterior cruciate ligament was found to have significant morphological differences in comparison with the other three tissues, including less compact collagen architecture, differences in cell nuclei phenotype and increased glycosaminoglycan and elastin content. Intra- and interobserver differences of histology scoring resulted in an average score 0.7, indicative of good agreement between observers. Statistically significant differences were also found in the extracellular matrix composition in terms of glycosaminoglycan and elastin content, being more prominent in the anterior cruciate ligament than in the other three tissues. A different distribution of several extracellular matrix proteins was also found between long digital extensor tendon and anterior cruciate ligament, with a significantly increased immunostaining of aggrecan and versican in the anterior cruciate ligament. These findings directly relate to the different functions of tendon and ligament and indicate that the intra-articular anterior cruciate ligament is subjected to more compressive forces, reflecting an adaptive response to normal or increased loads and resulting in different extracellular matrix composition and arrangement to protect the tissue from damage.

Diagnostic histochemistry of soft tissue lesions.

Histochemical methods (HM) were, at one time, extensively used in all facets of anatomic pathology, including analysis of soft tissue lesions. That situation has changed with the advent of other adjunctive procedures, but HM still do contribute meaningfully to the evaluation of several tumefactive conditions in the soft tissue. This brief review considers selected neoplastic, quasineoplastic, and pseudoneoplastic lesions in that category, with emphasis on their histochemical properties.

A novel application of periodic acid-Schiff (PAS) staining and fluorescence imaging for analysing tapetum and microspore development.

The precisely timed process of tapetum development and its degradation involving programmed cell death is an important molecular event during anther development. Through its degeneration, the tapetum not only provides nutritive substances to the developing microspores but also contributes to the pollen wall by way of sporopollenin, which is a complex mixture of biopolymers, containing long-chain fatty acids, phenylpropanoids, phenolics and traces of carotenoids. A number of dyes and staining methods have been used to visualize tapetal structure and its components by using light microscopy techniques, but none of these methods could differentially stain and thus distinguish tapetal cells from other cell types of anther wall. While analysing progression of tapetum development in different cell types in rice anthers, we discovered a unique property of periodic acid-Schiff (PAS) stain, which upon interaction with some specific component(s) in tapetal cells and developing microspores emits fluorescence at ~620 nm. In rice anthers, the PAS-associated fluorescence could be observed initially in tapetum and developing microspores, and subsequent to degeneration of tapetum, the fluorescence was found to emanate mainly from the pollen wall. We also show that PAS-dependent fluorescence in tapetal cells is distinct from the autofluorescence resulting from pollen wall components and is also not caused by interaction of PAS with pollen starch. Henceforth, this novel fluorescence property of PAS stain could prove to be a new tool in the toolkit of developmental biologists to analyse different aspects of tapetum development and its degeneration with little more ease and specificity.

Attenuation of oxidative stress and cardioprotective effects of zinc supplementation in experimental diabetic rats.

Oxidative stress plays a major role in the pathogenesis of diabetes mellitus, which further exacerbates damage of cardiac, hepatic and other tissues. We have recently reported that Zn supplementation beneficially modulates hyperglycaemia and hypoinsulinaemia, with attendant reduction of associated metabolic abnormalities in diabetic rats. The present study assessed the potential of Zn supplementation in modulating oxidative stress and cardioprotective effects in diabetic rats. Diabetes was induced in Wistar rats with streptozotocin, and groups of diabetic rats were treated with 5- and 10-fold dietary Zn interventions (0·19 and 0·38 g Zn/kg diet) for 6 weeks. The markers of oxidative stress, antioxidant enzyme activities and concentrations of antioxidant molecules, lipid profile, and expressions of fibrosis and pro-apoptotic factors in the cardiac tissue were particularly assessed. Supplemental Zn showed significant attenuation of diabetes-induced oxidative stress in terms of altered antioxidant enzyme activities and increased the concentrations of antioxidant molecules. Hypercholesterolaemia and hyperlipidaemia were also significantly countered by Zn supplementation. Along with attenuated oxidative stress, Zn supplementation also showed significant cardioprotective effects by altering the mRNA expressions of fibrosis and pro-apoptotic factors (by >50 %). The expression of lipid oxidative marker 4-hydroxy-2-nonenal (4-HNE) protein in cardiac tissue of diabetic animals was rectified (68 %) by Zn supplementation. Elevated cardiac and hepatic markers in circulation and pathological abnormalities in cardiac and hepatic tissue architecture of diabetic animals were ameliorated by dietary Zn intervention. The present study indicates that Zn supplementation can attenuate diabetes-induced oxidative stress in circulation as well as in cardiac and hepatic tissues.

Correlation between histopathologic features and likelihood of identifying superficial dermatophytosis with periodic acid Schiff-diastase staining: a cohort study.

BACKGROUND: Periodic acid Schiff-diastase (PAS-D) staining is used routinely in dermatopathology. Conventionally, parakeratosis or intracorneal neutrophils justify use of PAS-D stain to identify dermatophytosis. We aimed to investigate the histopathological features of superficial dermatophytosis. METHODS: Skin biopsies with PAS-D stain were retrospectively reviewed by a blinded dermatopathologist. Histopathologic findings in cases with dermatophytosis were compared with those without. RESULTS: We studied 110 cases, of which 63 were PAS-D positive for dermatophytes. Dermatophytes were identified on hematoxylin and eosin (H&E) staining in 49 (77.8%) of the PAS-D-positive cases. Diffuse compact orthokeratosis (79.37%, p < 0.001) and focal parakeratosis (65.08%, p = 0.03) were significantly more frequent in dermatophytosis. Intracorneal neutrophils and spongiosis were not significant findings. Fungal elements were frequently observed within compact orthokeratosis (62.0%) and parakeratosis (31.8%) but rarely within intracorneal pustules (12.7%). In some dermatophyte cases, intracorneal neutrophils (47.6%) or parakeratosis (17.5%) were absent. CONCLUSIONS: Compact orthokeratosis or focal parakeratosis justifies PAS-D staining, whereas the absence of intracorneal neutrophils or parakeratosis is insufficient to exclude dermatophytosis. Results indicate that having a low threshold to perform PAS-D stain is justified. PAS-D stain may be deferred until H&E slides have been evaluated.

Quantification of biofilm exopolysaccharides using an in situ assay with periodic acid-Schiff reagent.

A novel approach to the quantification of extracellular polysaccharides in miniaturized biofilms presenting a wide variety of extracellular matrices was developed. The assay used the periodic acid-Schiff reagent and was first calibrated on dextran and alginate solutions. Then it was implemented on 24-h and 48-h biofilms from three strains known to produce different exopolymeric substances (Pseudomonas aeruginosa, Bacillus licheniformis, Weissella confusa). The assay allowed quantification of the total exopolysaccharides, taking into account possible interferences due to cells or other main expolymers of the matrix (eDNA, proteins).

LC-MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences.

Glycogen synthesis is initiated by self-glucosylation of the glycosyltransferases glycogenin-1 and -2 that, in the presence of UDP-glucose, form both the first glucose-O-tyrosine linkage, and then stepwise add a series of α1,4-linked glucoses to a growing chain of variable length. Glycogen-1 and -2 coexist in liver glycogen preparations where the proteins are known to form homodimers, and they also have been shown to interact with each other. In order to study how glycogenin-1 and -2 interactions may influence each other's glucosylations we setup a cell-free expression system for in vitro production and glucosylation of glycogenin-1 and -2 in various combinations, and used a mass spectrometry based workflow for the characterization and quantitation of tryptic glycopeptides originating from glycogenin-1 and -2. The analysis revealed that the self-glucosylation endpoint was the incorporation of 4-8 glucose units on Tyr 195 of glycogenin-1, but only 0-4 glucose units on Tyr-228 of glycogenin-2. The glucosylation of glycogenin-2 was enhanced to 2-4 glucose units by the co-presence of enzymatically active glycogenin-1. Glycogenin-2 was, however, unable to glucosylate inactive glycogenin-1, at least not an enzymatically inactivated Thr83Met glycogenin-1 mutant, recently identified in a patient with severe glycogen depletion.

Comprehensive biometric, biochemical and histopathological assessment of nutrient deficiencies in gilthead sea bream fed semi-purified diets.

Seven isoproteic and isolipidic semi-purified diets were formulated to assess specific nutrient deficiencies in sulphur amino acids (SAA), n-3 long-chain PUFA (n-3 LC-PUFA), phospholipids (PL), P, minerals (Min) and vitamins (Vit). The control diet (CTRL) contained these essential nutrients in adequate amounts. Each diet was allocated to triplicate groups of juvenile gilthead sea bream fed to satiety over an 11-week feeding trial period. Weight gain of n-3 LC-PUFA, P-Vit and PL-Min-SAA groups was 50, 60-75 and 80-85 % of the CTRL group, respectively. Fat retention was decreased by all nutrient deficiencies except by the Min diet. Strong effects on N retention were found in n-3 LC-PUFA and P fish. Combined anaemia and increased blood respiratory burst were observed in n-3 LC-PUFA fish. Hypoproteinaemia was found in SAA, n-3 LC-PUFA, PL and Vit fish. Derangements of lipid metabolism were also a common disorder, but the lipodystrophic phenotype of P fish was different from that of other groups. Changes in plasma levels of electrolytes (Ca, phosphate), metabolites (creatinine, choline) and enzyme activities (alkaline phosphatase) were related to specific nutrient deficiencies in PL, P, Min or Vit fish, whereas changes in circulating levels of growth hormone and insulin-like growth factor I primarily reflected the intensity of the nutritional stressor. Histopathological scoring of the liver and intestine segments showed specific nutrient-mediated changes in lipid cell vacuolisation, inflammation of intestinal submucosa, as well as the distribution and number of intestinal goblet and rodlet cells. These results contribute to define the normal range of variation for selected biometric, biochemical, haematological and histochemical markers.