Acid Phosphatase

To determine the cytotoxicity of chemotherapy drugs, cell growth/viability was measured using an acid phosphatase assay; 1.5–3 × 10³ cells were seeded in flat-bottomed 96-well plates and incubated overnight prior to addition of drug. Chemotherapeutics were obtained from St Vincent’s University Hospital, Dublin, Ireland. Lapatinib was purchased from Sequoia. Temozolomide was obtained from the National Cancer Institute. Other inhibitors and modulators were obtained from Sigma. Drug-free controls were included in each assay. Plates were incubated for a further 5 (HCC1954, Malme-3M and HT144) or 7 days (H1299 and H460) at 37°C in a humidified atmosphere with 5% CO₂ and cell viability was determined using an acid phosphatase assay (97 (link)). Growth of drug-treated cells was calculated relative to control untreated cells in biological triplicate.

At the same time that the number of microorganisms was determined, i.e. on days 30 and 60 of the experiment in soil samples, from each repetition in three subsequent replications, the activity of dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, β-glucosidase and arylsulphatase was determined. The substrates used for the determination of the enzyme activity, as well as the units in which the activity of particular enzymes was expressed, are presented in Table 4. The activity of all enzymes, with the exception of catalase, was determined using a Perkin-Elmer Lambda 25 spectrophotometer (MA, USA). The activity of dehydrogenases was determined at a wavelength (λ) of 485 nm; the activity of urease, acid phosphatase and alkaline phosphatase at 410 nm; the activity of β-glucosidase at 400 nm; and the activity of arylsulphatase at 420 nm. The activity of catalase was determined based on the reaction of hydrogen peroxide decomposition using potassium permanganate.Table 4

Methods of determination of soil enzyme activity

Enzyme	Substrate	Product/unit	References
Deh—dehydrogenases (EC 1.1)	2,3,5-Triphenyl tetrazolium chloride (TTC)	Triphenyl fomazan (TFF), μmol kg−1 DM of soil h−1	Öhlinger (1996 )
Cat—catalase (EC 1.11.1.6)	H2O2—aqueous solution	O2, mol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Ure—urease (EC 3.5.1.5)	Urea—aqueous solution	N-NH4, mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Glu—β-glucosidase (EC 3.2.1.21)	4-Nitrophenyl-β-D-glucopyranoside (PNG)	4-Nitrophenol (PN), mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Pac—acid phosphatase (EC 3.1.3.2)	Disodium 4-nitrophenyl phosphate hexahydrate (PNP)	4-Nitrophenol (PN), mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Pal—alkaline phosphatase (EC 3.1.3.1)	Disodium 4-nitrophenyl phosphate hexahydrate (PNP)	4-Nitrophenol (PN), mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )
Aryl—aryosulphatase (EC 3.1.6.1)	Potassium-4-nitrophenylsulfate (PNS)	4-Nitrophenol (PN), mmol kg−1 DM of soil h−1	Alef and Nannipieri (1998 )

Canine EDTA blood samples containing mf (n=379) were collected between 2006 and 2011 by different parasitological laboratories in Europe and sent to Parasitus Ex e.V. in Niederkassel (lab 1). The samples were taken from dogs from Spain (73), Portugal (102), Greece (6), Italy (30), Romania (3), Hungary (141), Bulgaria (1), Turkey (2) and France (1); for 19 samples the dog origin was unknown. Dogs of both sexes and various age groups were sampled; exact age determination was impossible for most dogs due to their unknown history. The modified Knott’s technique was applied to concentrate and to detect mf. One ml of EDTA blood was mixed with 9 ml of 2% formalin in a 15 ml tube and centrifuged for 5 minutes at 500 × g. The supernatant was poured off, and 2 × 10 μl of the sediment was transferred to a slide and covered with a coverslip.
Morphometric analyses of the mf were conducted with standard diagnostic microscopes equipped with calibrated measuring eyepieces at a final magnification of 200–400 x. Body length and diameter, and the form of the front end and the tail of ten randomly selected mf were determined. To demonstrate the reliability of the measurements, a subset of 30 samples was repeatedly tested at the Institute of Parasitology, University of Zürich, Switzerland (lab. 2, D. immitis, D. repens and A. dracunculoides) and at the Department of Veterinary Science, University of Pisa, Italy (lab. 3, A. reconditum).
To estimate size variations in relation to time, microfilariae in two samples concentrated by the modified Knott’s technique were measured repeatedly after intervals of 30 minutes, 2 hours, 6 hours, 24 hours, 7 days, 18 days, 31 days, 45 days, and 105 days (storage of concentrated material at 4°C) by two independent operators in labs 1 and 2.
All samples had been identified to species level by demonstrating the characteristic acid phosphatase activity patterns
[5 (link)]. The Leucognost SP® kit was used according to the manufacturer’s recommendations to demonstrate acid phophatase activity patterns in the mf
[6 (link)]. The fourteen A. reconditum positive samples were additionally stained histochemically
[5 (link)], as no experience with Leucognost SP® exists for this species as yet.
Heartworm antigen detection tests were carried out on all blood samples (FASTest® Hw Ag.; MegaCor or DiroCHEK®, Synbiotics). To confirm species identification by acid phosphatase activity, 6 samples with D. immitis, 4 with D. repens, 7 with A. dracunculoides and 14 with A. reconditum were investigated by PCR
[7 (link)], and sequencing confirmed the species identification in all cases.

Almost 10 hetares of harvested sites of eucalypt plantation were divided into four equal blocks, and three planting patterns were randomly arranged within each block in July 2016. The first planting pattern (E) was the continuous planting of pure Eucalyptus. urograndis (hybrid strain of Eucalyptus urophylla and Eucalyptus grandis) plantations of the third generation at a density of 1,667 plants/ha. The second planting pattern (EC) was the creation of mixed plantations of E. urograndis and Cinnamomum camphora (mixed pattern: inter-row, mixed density: 1667 plants/ha). The third planting pattern (EH) was the creation of mixed plantations of E. urograndis and Castanopsis hystrix (mixed pattern: inter-row, mixed density: 1667 plants/ha). Simultaneously, four unmanaged first-generation E. urophylla plantations in Luogangling Forest Park were selected as controls (CK). Information on forestland preparation, seedling specifications of eucalypts and native trees, and later plantation tending can be found in Xu et al. (2022) (link).
Sixteen mixed topsoil samples in the 10-cm layer were collected in December 2019 by removing the humus and litterfall from four different planting patterns. Soil samples for fungal community structure analysis were preserved with dry ice in centrifuge tubes and transferred to a-80°C freezer as soon as possible. Other soil samples for analyses of soil chemical properties and enzyme activities were stored in a portable refrigerator at 4°C.
The pH of each sample was determined with an electronic pH meter (soil: water, 1:2.5). Soil OM was determined by the potassium dichromate-sulfate colorimetric method (Sims and Haby, 1971 (link)). Total nitrogen (TN) and total phosphorus (TP) were measured with the Kjeldahl method (Tsiknia et al., 2014 (link)) and sodium hydroxide fusion-molybdenum antimony colorimetric method (Liu H. et al., 2017 (link)), respectively. Nitrate nitrogen (NO¯ 3_N) was determined by 2 mol·L⁻¹ KCl leaching-indophenol blue colorimetric method and ammonium nitrogen (NH+ 4_N) was determined by UV spectrophotometry (Lu, 1999 ). Available phosphorus (AP) was measured by the hydrochloric acid-ammonium fluoride extraction-molybdenum antimony colorimetric method (Lu, 1999 ). Soil available zinc (AZn) and available calcium (ACa) were measured by hydrochloric acid extract, atomic absorption spectrophotometry and ammonium acetate exchange, atomic absorption spectrophotometry, respectively (Liu J. et al., 2017 (link)). For soil enzyme activities, acid phosphatase (ACP) was determined by Phenylphosphonium-4-amino-antipyrine colorimetric method (Guan, 1986 ), urease (URE) by alkaline dish diffusion-HCL titration method (Guan, 1986 ), and invertase (INV) by 3,5-Dinitrosalicylic acid colorimetric method (Lu, 1999 ).

After culturing the cells were fixed with 4% PFA in PBS for 10 min at RT. The actin cytoskeleton was stained with Alexa 488-conjugated phalloidin (200 U/ml stock diluted 1:100 in PBS; Invitrogen Europe, Paisley, UK) for 20 min at + 37 °C. Nuclei were stained with Hoechst 33258 (1 mg/ml stock diluted 1:800 in PBS; Sigma-Aldrich) for 10 min at room temperature (RT). Staining for osteoclast-specific enzyme TRACP was carried out with a commercial acid phosphatase leukocyte kit (Sigma-Aldrich) for 20 min at + 37 °C. The samples were mounted in 70% glycerol-PBS and viewed in a Zeiss Axio Scope.A1 fluorescence microscope (Oberkochen, Germany) and EC Plan Neofluar 20 × objective. Multinuclear cells with three or more nuclei were counted from each bone slice from five randomly chosen microscope fields, bone slice n = 4–6. The number of nuclei per cell were counted from 5 multinuclear cells from 5 randomly chosen areas. Images were taken with Nikon Eclipse E600 fluorescence microscope using Plan 20 ×/0.5 objective (Tokyo, Japan), QImaging MicroPublisher 5.0 RTV camera and QCapture 2.90.1 software (QImaging, Surrey, Canada). Confocal images were taken with Leica TCS SP8 confocal with a DMI8 microscope using LAS X 3.5.2 acquisition software. The objective used was an HC PL APO CS2 20 ×/0.75 DRY. Samples were imaged with 488 nm and 405 nm solid-state lasers; the pinhole was set to Airy 1 and scan speed to 600 Hz.