Atomizers

Immature C. sinensis specimens were purchased from local markets from the Hualong (located at 36°13’N, 102°19’E) and Yushu areas (located at 33°01’N, 96°48’E) of Qinghai Province of China (3800–4600 m’ altitude) in mid-May and characterized by a plump caterpillar body and very short stroma (1.0–2.0 cm) [19 , 27 ]. Mature C. sinensis specimens were collected in mid-June and characterized by a plump caterpillar body and long stroma (>5.0 cm) and by the formation of an expanded fertile portion close to the stromal tip, which was densely covered with ascocarps (Fig 1). Governmental permission was not required for C. sinensis purchases in local markets, and the collections of C. sinensis specimens from sales by local farmers fall under the governmental regulations for traditional Chinese herbal products.
The specimens were washed thoroughly on site in running water with gentle brushing, soaked in 0.1% mercuric chloride for 10 min for surface sterilization and washed 3 times with sterile water. The thoroughly cleaned specimens were immediately frozen in liquid nitrogen on site and kept frozen during transportation to the laboratory and during storage prior to further processing [19 , 27 ].
Some of the mature C. sinensis specimens were harvested along with the outer mycelial cortices and soil surrounding the caterpillar body and replanted in paper cups in soil obtained from C. sinensis production areas (Fig 1A) and were cultivated in our laboratory (altitude 2,200 m) in Xining City, Qinghai Province of China [67 , 68 ]. Because of the phototropism of natural C. sinensis, we kept the windows fully open, allowing sufficient sunshine and a natural plateau breeze blowing over the cultivated specimens in the paper cups. The room temperature was maintained naturally, fluctuating with the lowest temperature at 18–19°C during the night and the highest temperature at 22–23°C in the early afternoon. The humidity of our laboratory was maintained by spraying of water using an atomizer twice a day in the morning and evening.
Fully ejected ascospores of C. sinensis were collected using double layers of autoclaved weighing paper (Fig 1B). During massive ascospore ejection, numerous ascospores adhered to the outer surface of asci, as shown in Fig 1C after removing the upper layer of autoclaved weighing papers for collection of the fully ejected ascospores, and failed to be brushed away using an autoclaved brush; hence, these ascospores were instead gently scratched off using a disinfected inoculation shovel or ring and referred to as semiejected ascospores.
The 2 types of ascospores were cleaned by 2 washes with 10% and 20% sucrose solutions and 10-min centrifugation at 1,000 rpm (desktop centrifuge, Eppendorf, Germany); the supernatant was discarded after each centrifugation. The pellets (ascospores) were subsequently washed with 50% sucrose solution and centrifuged for 30 min, and the ascospores that floated on the liquid were collected [67 ]. The fully and semiejected ascospores were stored in a -80°C freezer prior to further processing.

It was decided to collect a single spot urine sample and store it at ≤-20°C for long-term or short-term analysis stored at 2–8°C before analysis. Inductively coupled plasma mass spectrometry (ICP-MS) was employed to determine nickel levels in urine, which is a susceptible technique that can measure multiple elements at low concentrations. To be brief, ICPs operate with argon flows passing through an atomizer and spray chamber to process urine samples. The sample vaporizes at a high temperature, dissociates the ionized gas, and then the ions reach the ion detector. Finally, the isotope ratio of the elements is measured. A urinary nickel concentration of 0.31 mg/L is considered a detection limit of detection. More detailed laboratory procedure manuals are shown on the NHANES's website (27 ).
This study measured the weight, height, and WC of adults 18 and older using standardized methods. Physical examination measured BMI and WC. BMI is calculated by dividing the square of a person's weight (in kilograms) by their height (in meters), and precise measurements could well be acquired using standard digital scales and rulers. Medical professionals measured the subjects' WC with a flexible ruler. According to the WHO-recommended measurement method, the subject's feet were separated by 25–30 cm. The measurer placed the measuring tape around the abdomen in a circle at the midpoint of the line connecting the anterior superior iliac crest and the lower border of the 12th rib, close to the soft tissue, but without compression, and measured at the end of exhalation and before inspiration. When the WC and BMI are both normal, it is not obesity; when the BMI is normal, but the WC of men is ≥94 cm, and that of women is ≥80 cm, it is central obesity; when the WC is standard, but the BMI is ≥30 kg/m², it is defined as peripheral obesity; when the BMI and WC are both above normal, it is defined as mixed obesity (28 (link), 29 ).