Unraveling aerobic cultivable cellulolytic microorganisms within the gastrointestinal tract of sheep (Ovis aries) and their evaluation for cellulose biodegradation

Anaerobic cellulolytic microbes in gastrointestinal tract (GT) of ruminants have been well-documented, however, knowledge of aerobic microbes with cellulolytic activities in ruminant GT is comparably limited. Here, we unraveled aerobic cultivable cellulolytic microbes in GT of Ujimqin sheep (Ovis aries) and evaluated the cellulolytic potential of promising isolates. Twenty-two strains were found to possess cellulose degrading potential by Congo-red staining and phylogenetic analysis of the 16S rDNA/ITS sequence revealed that all strains belonged to nine genera, i.e., Bacillus, Streptomyces, Pseudomonas, Lactobacillus, Brachybacterium, Sanguibacter, Rhizobium, Fusarium, and Aspergillus. Strains with high cellulolytic activities were selected to further evaluate the various enzyme activities on lignocellulosic alfalfa hay (Medicago sativa). Among them, isolate Bacillus subtilis RE2510 showed the highest potential of cellulose degradation considering the high endoglucanase (0.1478 ± 0.0014 IU ml-1), exoglucanase (0.1735 ± 0.0012 IU ml-1) and β-glucosidase (0.3817 ± 0.0031 IU ml-1) after 10-day incubation with alfalfa hay. A significant destruction effect of the cellulose structure and the attachment of B. subtilis RE2510 to the hay were also revealed by using scanning electron microscope. This study expands our knowledge of aerobic cellulolytic isolates from GT of sheep and also highlights their potential application as microbial additive in the aerobic process of cellulose bioconversion.

AA-amyloidosis in captive northern tree shrews (Tupaia belangeri)

A high prevalence of AA-amyloidosis was identified in a breeding colony of northern tree shrews ( Tupaia belangeri) in a retrospective analysis, with amyloid deposits in different organs being found in 26/36 individuals (72%). Amyloid deposits, confirmed by Congo red staining, were detected in kidneys, intestines, skin, and lymph nodes, characteristic of systemic amyloidosis. Immunohistochemically, the deposited amyloid was intensely positive with anti-AA-antibody (clone mc4), suggesting AA-amyloidosis. The kidneys were predominantly affected (80%), where amyloid deposits ranged from mild to severe and was predominantly located in the renal medulla. In addition, many kidneys contained numerous cysts with atrophy of the renal parenchyma. There was no significant association between concurrent neoplastic or inflammatory processes and amyloidosis. The lack of distinctive predisposing factors suggests a general susceptibility of captive T. belangeri to develop amyloidosis. Clinical and laboratory findings of a female individual with pronounced kidney alterations were indicative of renal failure. The observed tissue tropism with pronounced kidney alterations, corresponding renal dysfunction, and an overall high prevalence suggests amyloidosis as an important disease in captive tree shrews.

Macroglossia reveals multiple myeloma

We present an eighty-year old man with a one year history of progressive macroglossia, dysphagia and loss of weight. He had a medical history of arterial hypertension and prostatic hypertrophy which he had under good therapeutic control. The entire tongue was swollen, had hard solidity and was slightly painful upon palpation. A tongue biopsy revealed an amyloid deposition as it coloured bright orange-red on Congo Red staining and lead us subsequently to the diagnosis of amyloidosis; then a bone marrow biopsy confirmed the diagnosis of multiple myeloma. The case was further evaluated by a multidisciplinary team who considered it appropriate to start a lowdose melphalan treatment combined with supportive care. When macroglossia in the tongue is confirmed to be amyloidosis the differential diagnosis should include systemic amyloidosis deposition and multiple myeloma.

Exploring cellulolytic microorganisms from coffee industry by-products and their enzyme properties

Cellulolytic microorganism has immense potential due to their cellulase production, enzyme complexity and widespread habitat of life. This study was conducted to obtain microbial cellulase with vast industrial applicability from the coffee industry by-product in East Java, Indonesia. Fifty-four isolates with significant clear zone formation were obtained by Congo red staining in CMC agar plates. Eighteen bacteria, two yeasts and two moulds with high cellulolytic index were subjected to protein content determination as well as reducing sugar analysis in various conditions such as pH, temperature, addition of metal ions, surfactant and inhibitor agent. The specific activity measurements of all the crude enzymes result in the highest value of cellulase activity produced by isolate C12 which was 0.401 ± 0.018 U/mg. This enzyme activity was known to be optimum at 50°C and pH 9. It was also stimulated by K+, Na+, Mg2+, Fe3+, and SDS. However, the enzyme activity was inhibited by EDTA at 10 mM concentration. The use of coffee industry by-products as the source of cellulolytic microorganisms offers a promising approach for its various types of indigenous microorganisms and their unique property of cellulase produced that is useful for industrial application.

Diagnosis of Primary Cutaneous Amyloidosis by Rapid 4,6-Diamidino-2-Phenylindole Staining

<b><i>Background:</i></b> Quick and accurate diagnosis of primary cutaneous amyloidosis (PCA) may be difficult because its symptoms are often subtle and nonspecific. <b><i>Objective:</i></b> We sought to review the literature on the roles of various staining methods in the diagnosis of amyloidosis and demonstrate added benefits of using rapid 4,6-diamidino-2-phenylindole (DAPI) staining in the diagnosis of PCA. <b><i>Methods:</i></b> Three groups of cases, namely, PCA, neurodermatitis, and prurigo nodularis, were retrieved from a computerized pathology database for study, and their paraffin-embedded tissue blocks were cut following standard procedures. The tissue sections were stained with three stains: hematoxylin-eosin (HE), Congo red, and DAPI stains, and examined under the microscope to compare the staining patterns of these three methods. We also performed amyloid keratin and apolipoprotein E (APOE) staining on the sections of PCA in order to further support our conclusion. The PCA sections were read by junior and senior dermatopathologists for comparison. <b><i>Results:</i></b> The sensitivity of DAPI staining for PCA was significantly higher than that of Congo red staining and HE staining (<i>p</i> &#x3c; 0.001). This statement holds true whether the experiment was grouped in one sample or was divided into groups of junior and senior dermatopathologists (<i>p</i> &#x3c; 0.001). The DAPI-positive staining areas, except for the nuclei, were consistent with the amyloid deposition areas. In this study, DAPI staining had a sensitivity of 98.6% and a specificity of 100%. <b><i>Conclusion:</i></b> DAPI staining could serve as a useful technique to establish the diagnosis of PCA, and its high efficacy in diagnosing PCA makes it less dependent on the experience levels of the evaluators. Additionally, the binding of DAPI to the A-T-rich sequence of double-stranded DNA suggests that amyloid may contain DNA or a similarly structured nucleic acid.

Amyloidosis in major body organs examined during autopsies at Mbarara Regional Referral Hospital

BackgroundAmyloidosis is an infrequent disease that occurs when an abnormal protein, called amyloid, deposits in body organs and disrupts their normal function. Amyloid is not normally found in the body but it can be formed from several different types of protein. Commonly affected organs include the heart, kidneys, gastrointestinal (GI) tract/liver or the peripheral or autonomic nervous system (NS). Amyloidosis can lead to diseases such as Alzheimer’s disease, spongiform encephalopathies and diabetes mellitus type 2. Main objectiveTo establish the prevalence of amyloidosis in major body organs examined during autopsies at Mbarara regional referral hospital and to correlate cause of death with amyloidosis. MethodsBrain, heart, kidney, and liver samples were obtained from 23 bodies during autopsies at Mbarara regional referral hospital which were then processed to produce 2 slides per tissue of each of the above organ samples. These slides were then stained using H&E and Congo red staining protocols and then examined for amyloidosis under the light microscope. ResultsThe mean age of the bodies was 61.26 of which 5 (21.7%) were female while 18 (78.3%) were male. 3 bodies (13.0%) tested positive for amyloidosis in the kidney; 2 males (8.70) and 1 female (4.35). All the other organs tested negative. ConclusionIn our study, body organ investigation revealed amyloid in the kidney of 3 bodies. The prevalence of amyloidosis was 13% therefore amyloidosis diagnosis ought to be put into consideration at Mbarara Regional Referral hospital.

Combined Subcutaneous Fat Aspirate and Skin Tru-Cut Biopsy for Amyloid Screening in Patients with Suspected Systemic Amyloidosis

Screening for systemic amyloidosis is typically carried out with abdominal fat aspirates with varying reported sensitivities. Fat aspirates are preferred for use in primary screening instead of organ biopsies as they are less invasive and thereby minimize the potential risk of complications. At Odense Amyloidosis Center, we performed a prospective study on whether the combined use of fat aspirate and tru-cut skin biopsy could increase the diagnostic sensitivity. Both fat aspirates and skin biopsies were screened with Congo Red staining, and positive biopsies were subsequently subtyped using immunoelectron microscopy and mass spectrometry. Seventy-six patients were included. In total, 24 patients had systemic amyloidosis (11 AL, 12 wtATTR, 1 AA), and 6 patients had localized amyloidosis. Combined fat aspirate and skin biopsy were Congo Red-positive in 15 patients (overall sensitivity (OS) 62.5%). Fat aspirates were positive in 14 patients (OS 58.3%), and the skin biopsy was positive in 5 patients (OS 20.8%). In only one patient did the skin biopsy add extra diagnostic information. The sensitivity differed between AL and ATTR amyloidosis—81.8% and 41.7%, respectively. Using skin biopsy as the only screening method is not recommended.

Bioethanol Production of Cellulase Producing Bacteria from Soils of Agrowaste Field

With decades of studies on cellulose bioconversion, cellulases have been playing an important role in producing fermentable sugars from lignocellulosic biomass. Copious microorganisms that are able to degrade cellulose have been isolated and identified. The present study has been undertaken to isolate and screen the cellulase producing bacteria from soils of agrowaste field. Cellulase production has been qualitatively analyzed in carboxy methylcellulose (CMC) agar medium after congo red staining and NaCl treatment by interpretation with zones around the potent colonies. Out of the seven isolates, only two showed cellulase production. The morphogical and molecular characterization revealed its identity as Escherichia coli and Staphylococcus aureus. The potential of organisms for bioethanol production has been investigated using two substrates, namely, paper and leaves by subjecting with a pre-treatment process using acid hydrolysis to remove lignin which acts as physical barrier to cellulolytic enzymes. Ethanolic fermentation was done using Saccharomyces cerevisiae for 24-48 h and then the bioethanol produced was qualitatively proved by iodoform assay. These finding proves that ethanol can be made from the agricultural waste and the process is recommended as a means of generating wealth from waste.

Nephrotic Syndrome Following Resection of an Adrenal Incidentaloma:A Case Report

A 69 year old man had a 5 cm right adrenal lesion discovered incidentally while being investigated for a deterioration in previously well-controlled hypertension. Routine investigations including serum albumin were normal. Further investigation confirmed a non-functioning adrenal lesion. MRI revealed a ‘non-fat-containing T1 hyperintense indeterminate adrenal lesion with speckling of T2 hyperintensity, not typical for adenoma, hyperplasia, myelolipoma, haemangioma or pheochromocytoma’. An uncomplicated laparoscopic adrenalectomy was performed. Histology revealed a 118 g adrenal neoplasm, modified Weiss score 0, with abundant hyaline deposits.3 months later the patient complained of peripheral oedema. Investigations revealed a serum albumin of 24 g/L and 14 g of proteinuria in 24 hours. Serum protein electrophoresis revealed a monoclonal IgA type lambda band. Renal biopsy revealed amorphous material displaying apple green birefringence on staining with Congo Red, which stained with antibodies to lambda light chains, confirming AL amyloid. Therefore the patient’s resected adrenal specimen was retrieved and stained with Congo Red, revealing apple green birefringence in the walls of the blood vessels, confirming the presence of amyloidosis. Although adrenal gland involvement in secondary amyloidosis is common, adrenal involvement in primary amyloidosis is less well described. This case illustrates the indolent nature of primary amyloidosis, prior to the development of often catastrophic symptoms. Consideration should be given to Congo Red staining of resected pathologic specimens containing hyaline deposition, to potentially allow for earlier recognition of this devastating disease. A pathophysiologic link between the patient’s incidentaloma, adrenalectomy, and onset of nephrotic syndrome remains a matter for conjecture.

Systemic light-chain amyloidosis incidentally diagnosed after subtotal parathyroidectomy and thyroid lobectomy

A 74-year-old woman with a history of primary hyperparathyroidism, thyroid nodules, atrial fibrillation and pacemaker placement for sick sinus syndrome presented with fatigue, constipation and persistent lower extremity oedema. She underwent subtotal parathyroidectomy and left thyroid lobectomy. Histopathology revealed amyloidosis affecting the thyroidand parathyroids confirmed by Congo Red Staining with Mayo Clinic subtyping of light chain kappa-type amyloidosis. She was found to have combined systolic and diastolic cardiac dysfunction, carpal tunnel neuropathy and pre-diabetes suggestive of systemic amyloidosis with involvement of the heart, nerves and pancreas. Congo red stain was positive for amyloidosis on bone marrow biopsy suggestive of a diagnosis of systemic amyloidosis. She was treated with daratumumab with good clinical response. This case illustrates the necessity of considering systemic amyloidosis in patients with incidentally discovered diffuse amyloid deposits on biopsy of an endocrine organ, as endocrine effects are a rare but likely underdiagnosed consequence of systemic amyloidosis.