We report a new set of ultra-sensitive Ca 2+ indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca 2+ -sensing domain of a genetically encoded Ca 2+ indicator, YC2.60 or YC3.60. Their high Ca 2+ affinities (K d = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca 2+ transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multi-cellular networks.3To decipher the principles of information processing in multi-cellular networks, such as a brain or developing embryo, it is essential to record cellular activity with fine We therefore generated YCs with a longer linker, containing 3 to 8 amino acids (designated 3 to 8) (Fig. 1a). The Ca 2+ affinity gradually increased as the linker was elongated and we named the resulting series of sensors, high-affinity yellow cameleons or "YC-Nano". For the 3 (Gly-Gly-Ser) linker the K d was 50 nM (YC-Nano50) and for the 4 (Gly-Gly-Gly-Ser) linker the K d was 30 nM (YC-Nano30). YCs with a 5 to 8 5 linker also had a higher affinity than YC2.60. The lowest K d (= 15 nM) was achieved with a 5 linker (Gly-Gly-Gly-Gly-Ser) (YC-Nano15); this indicator had the highest affinity of any GECI reported so far 11 (Fig. 1b, Supplementary Table 1 and Supplementary Fig. 1). Linker elongation was also effective for YC3.60, yielding YC-Nano140 (4; Gly-Gly-Gly-Ser, K d = 140 nM) and YC-Nano65 (5;Gly-Gly-Gly-Gly-Ser, K d = 65 nM) (Fig. 1b, Supplementary Fig. 1 and Supplementary Table 1).Kinetic measurement by stopped-flow fluorometry of YC-Nano140 and YC3.60 revealed that only the rate constant for the on reaction was increased, while that of the off reaction remained unchanged (Supplementary Fig. 2 Fig. 3). Supplementary Fig. 4). To verify the advantages of YC-Stimulation with 10 M cAMP yielded large FRET signal changes, assessed by ratiometric wide-field imaging of aggregation-competent cells (Fig. 1c). The YFP/CFP for the YC-Nano15-and YC2.60-expressing cells changed from 5.0 to 9.5 (R = 4.5) and 2.0 to 6.2 (R = 4.2), respectively. We next determined the amplitude of the Ca The increased signal strength achieved by optimizing the K d also allowed us to perform Ca 2+ imaging on a large spatial scale. The field of view for imaging could be expanded to a millimeter-sized network that included 100,000-Dictyostelium cells, in which the aggregation wave was clearly visible as a rotating spiral (Supplementary Fig. 5, Supplementary Video 2, R Nano15 = 0.6), indicating that YC-Nano15, unlike YC2.60, was useful for detecting multi-cellular network activity in self-organized signaling dynamics (R YC2.60 = 0.15, Supplementary Fig. 6, Supplementary Video 3).We next tested the performance of YC-Nano in a neuronal system (Supplementary Note 3 and Supplementary Figure 7). For this, we examined YC-Nano's sensitivity for the subtle Ca 2+ transient triggered by a single action potential (AP). YC-Nano15 and 7 YC3...

IMPORTANCE Patients with cancer and health care workers (HCWs) are at high risk of SARS-CoV-2 infection. Assessing the antibody status of patients with cancer and HCWs can help understand the spread of COVID-19 in cancer care. OBJECTIVE To evaluate serum SARS-CoV-2 antibody status in patients with cancer and HCWs during the COVID-19 pandemic in Japan. DESIGN, SETTING, AND PARTICIPANTS Participants were enrolled for this prospective cross-sectional study between August 3 and October 30, 2020, from 2 comprehensive cancer centers in the epidemic area around Tokyo, Japan. Patients with cancer aged 16 years or older and employees were enrolled. Participants with suspected COVID-19 infection at the time of enrollment were excluded. EXPOSURES Cancer of any type and cancer treatment, including chemotherapy, surgery, immune checkpoint inhibitors, radiotherapy, and targeted molecular therapy. MAIN OUTCOMES AND MEASURES Seroprevalence and antibody levels in patients with cancer and HCWs. Seropositivity was defined as positivity to nucleocapsid IgG (N-IgG) and/or spike IgG (S-IgG). Serum levels of SARS-CoV-2 IgM and IgG antibodies against the nucleocapsid and spike proteins were measured by chemiluminescent enzyme immunoassay. RESULTS A total of 500 patients with cancer (median age, 62.5 years [range, 21-88 years]; 265 men [55.4%]) and 1190 HCWs (median age, 40 years [range, 20-70 years]; 382 men [25.4%]) were enrolled.In patients with cancer, 489 (97.8%) had solid tumors, and 355 (71.0%) had received anticancer treatment within 1 month. Among HCWs, 385 (32.3%) were nurses or assistant nurses, 266 (22.4%) were administrative officers, 197 (16.6%) were researchers, 179 (15.0%) were physicians, 113 (9.5%) were technicians, and 50 (4.2%) were pharmacists. The seroprevalence was 1.0% (95% CI, 0.33%-2.32%) in patients and 0.67% (95% CI, 0.29%-1.32%) in HCWs (P = .48). However, the N-IgG and S-IgG antibody levels were significantly lower in patients than in HCWs (N-IgG: β, −0.38; 95% CI, −0.55 to −0.21; P < .001; and S-IgG: β, −0.39; 95% CI, −0.54 to −0.23; P < .001). Additionally, among patients, N-IgG levels were significantly lower in those who received chemotherapy than in those who did not (median N-IgG levels, 0.1 [interquartile range (IQR), 0-0.3] vs 0.1 [IQR, 0-0.4], P = .04). In contrast, N-IgG and S-IgG levels were significantly higher in patients who received immune checkpoint inhibitors than in those who did not (median N-

Effects of different ageing methods on colour, yield, oxidation and sensory qualities of Australian beef loins consumed in Australia and Japan

p16 expression was a reliable prognostic biomarker regardless of human papillomavirus status.

1Milk produced by the grazing system, referred to as "grazing milk" contains many 2 components required for human health. The milk fatty acid (FA) profile is strongly 3 associated with the diet on the farms. In the present study, based on the FA profile 4 of farmer's bulk milk, we determined how to discriminate between milk produced 5 on grazing and on a confinement system. A field survey was conducted four times were collected and the FA profile of milk was measured. Milk produced during the 9 grazing season contained less C16:0 and cis-9 C16:0, and more C18:0, cis-9 C18:1, 10 trans-11 C18:1, cis-9,12 C18:2, cis-9,trans-11 C18:2 and cis-9,12,15 C18:3 than milk 11 produced during the confinement season. Discrimination analysis using 16 FA 12 revealed that almost all milk samples were discriminated correctly (confinement 13 season: 90% correct and 10% borderline, grazing season: 88% correct, 9% 14 borderline and 3% incorrect). For farmers that were categorized incorrectly and 15 were considered borderline in the grazing season, the dependency on pasture was 16 low compared with that for farmers discriminated correctly. Therefore, to claim 17 "grazing milk", a high dependency on pasture is required for grazing dairy farmers.

Purpose: Many articles have discussed the clinical features of previously untreated parotid cancer, but the clinical characteristics and treatment of recurrent parotid cancer have not yet been fully described. Materials: We retrospectively reviewed 20 patients with recurrent parotid cancer and analyzed the therapeutic strategies and the prognostic factors. Results: Twelve patients (60%) underwent definitive surgery, including 3 who underwent skull base surgery. The 5-year overall survival (OS) and 5-year disease-free survival (DFS) in the surgery group were 66.7 and 64.1%. In the definitive surgery group, the presence of lymph node metastasis and high-grade malignant histopathology were associated with a poor prognosis (p < 0.01). On the other hand, the presence of facial palsy at presentation, the surgical margin, the time of relapse and the T stage did not affect the DFS in our series. Conclusions: The results suggest that aggressive definitive surgery may be justified for the treatment of recurrent parotid cancer. The presence of lymph node metastasis and the histopathological malignancy grade are poor prognostic factors for OS and DFS.

Background: Lymph node metastasis with extranodal extension represents one of the most important adverse prognostic factors for survival in patients with head and neck squamous cell carcinoma. We propose that extranodal extension occurs to differing extents. The aim of this study was to determine the prognostic significance of extranodal extension in patients with head and neck squamous cell carcinoma. Methods: Two hundred and ninety-eight patients with head and neck squamous cell carcinoma who underwent surgical resection and neck dissection were included. Cervical lymph nodes were classified into four categories: (i) pathological N negative, (ii) extranodal extension negative, (iii) non-surgical extranodal extension and (iv) surgical extranodal extension. Results: Lymph node metastases were detected in 67.1% of laryngeal/hypopharyngeal cancer patients and 52.7% of oral cancer patients. The 3-year disease-specific survival rates for patients in the pathological N negative, extranodal extension negative, non-surgical extranodal extension and surgical extranodal extension groups were 90.9%, 79.6%, 63.8% and 48.3%, respectively. In laryngeal/hypopharyngeal cancer patients, surgical extranodal extension was associated with a significantly poorer disease-specific survival than a pathological N negative, extranodal extension negative or non-surgical extranodal extension status. In oral cancer patients, no significant differences were observed between the non-surgical and surgical extranodal extension groups. However, non-surgical extranodal extension was associated with a poorer disease-specific survival than a pathological N negative or extranodal extension negative status. Conclusions: Surgical extranodal extension was a poor prognostic factor in patients with head and neck squamous cell carcinoma. The prognostic significance of surgical extranodal extension differed between laryngeal/hypopharyngeal and oral cancer patients. The clinical significance of surgical extranodal extension was much greater for patients with laryngeal/hypopharyngeal cancer than oral cancer.

Purpose: Head and neck squamous cell carcinoma of an unknown primary site (HNSCCUP) is a heterogeneous group of tumors that includes the human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma. To investigate the relationship between HNSCCUP and HPV, we reviewed p16 overexpression and HPV DNA in lymph node metastases and examined their correlation with the primary site and clinical features. Materials and Methods: Thirty-three patients with HNSCCUP were retrospectively studied. Dissected neck metastases were analyzed for p16 overexpression by immunohistochemistry, and the presence of HPV DNA was investigated by in situ hybridization. Results: Of the 33 patients, 8 (24%) exhibited p16 overexpression. p16-positive lymph node metastases contained significantly more HPV DNA and were most frequently associated with occult primary lesions in the oropharynx and a favorable prognosis. Patients with a lower alcohol consumption, only level II/III metastasis, and cystic lymph node metastasis tended to have p16 overexpression. Conclusions: This is the first report on the relationship of HNSCCUP with p16 and HPV DNA status in Asian patients. In total, 24% of the HNSCCUP patients were p16 positive. p16 overexpression in neck metastasis was predictive of both an occult primary lesion in the oropharynx and an association with HPV infection. Alcohol consumption, location, and features of neck metastasis were correlated with p16 expression.