Insight into the aroma quality of 'Callista' cultivar of hop (Humulus lupulus L.): Impact of harvest timing, year, and location.

Hops (Humulus lupulus L.) are essential ingredients in brewing, contributing to beer's flavor, aroma, and stability. This study pioneers an in-depth analysis of the 'Callista' cultivar, aiming to unravel how harvest timing, annual variations, and cultivation location synergistically influence its molecular profile, sensory perception, and biochemistry. Leveraging high-performance liquid chromatography and gas chromatography-mass spectrometry-olfactometry, we identified significant year-to-year and location-based fluctuations in bitter acids-the quintessential aroma constituents in hops. Our comprehensive aroma profiling discerned 55 volatile compounds, marking the first-ever sensory detection of 2-butanone in hops, with its presence showing remarkable interannual variability. This study showed significant differences among the three years tested, whereas hops were perceived "fruitier" and more "citrusy" in 2021, even though the bitter acid and aroma analysis showed that 2022 sticks out due to extremely high lupulone values up to 10% dry cone weight and 78% ß-myrcene in the oil fraction compared to 60% and 45% in 2020 and 2021, respectively. Molecular analysis of key enzymes involved in hop aroma biosynthesis revealed no significant associations with location, but a strong diurnal pattern for all genes. The results indicated that especially the hot temperatures of 2022 may have induced significant changes of cone quality, while 2021 was more interesting from the sensory evaluations, which may justify the usage of viticultural terms such as "vintage" for hop marketing. These findings contribute to a better understanding of the factors influencing hop aroma and quality.

Validation of the pretreatment derived neutrophil-lymphocyte ratio as a prognostic factor in a European cohort of patients with upper tract urothelial carcinoma.

BACKGROUND: The value of a combined index of neutrophil and white cell counts, named derived neutrophil-lymphocyte ratio (dNLR), has recently been proposed as a prognosticator of survival in various cancer types. We investigated the prognostic role of the dNLR in a large European cohort of patients with upper tract urothelial carcinoma (UTUC). METHODS: Data from 171 non-metastatic UTUC patients, operated between 1990 and 2012 at a single tertiary academic centre, were evaluated retrospectively. Cancer-specific- (CSS) as well as overall survival (OS) were assessed using the Kaplan-Meier method. To evaluate the independent prognostic significance of the dNLR, multivariate proportional Cox-regression models were applied. Additionally, the influence of the dNLR on the predictive accuracy of the multivariate model was further determined by Harrell's concordance index (c-index). RESULTS: The median follow-up period was 31 months. An increased dNLR was statistically significantly associated with shorter CSS (log-rank P=0.004), as well as with shorter OS (log-rank P=0.002). Multivariate analysis identified dNLR as an independent predictor for CSS (hazard ratio, HR=1.16, 95% confidence interval, CI=1.01-1.35, P=0.045), as well as for OS (HR=1.21, 95% CI=1.09-1.34, P<0.001). The estimated c-index of the multivariate model for OS was 0.68 without dNLR and 0.73 when dNLR was added. CONCLUSIONS: Patients with a high pretreatment dNLR could be predicted to show subsequently higher cancer-specific- as well as overall mortality after surgery for UTUC compared with those with a low pretreatment dNLR. Thus, this combined index should be considered as a potential prognostic biomarker in future.

High plasma fibrinogen level represents an independent negative prognostic factor regarding cancer-specific, metastasis-free, as well as overall survival in a European cohort of non-metastatic renal cell carcinoma patients.

BACKGROUND: In recent years, plasma fibrinogen has been ascribed an important role in the pathophysiology of tumour cell invasion and metastases. A relatively small-scale study has indicated that plasma fibrinogen levels may serve as a prognostic factor for predicting clinical outcomes in non-metastatic renal cell carcinoma (RCC) patients. METHODS: Data from 994 consecutive non-metastatic RCC patients, operated between 2000 and 2010 at a single, tertiary academic centre, were evaluated. Analyses of plasma fibrinogen levels were performed one day before the surgical interventions. Patients were categorised using a cut-off value of 466 mg dl⁻¹ according to a calculation by receiver-operating curve analysis. Cancer-specific (CSS), metastasis-free (MFS), as well as overall survival (OS) were assessed using the Kaplan-Meier method. To evaluate the independent prognostic impact of plasma fibrinogen level, a multivariable Cox regression model was performed for all three different endpoints. RESULTS: High plasma fibrinogen levels were associated with various well-established prognostic factors, including age, advanced tumour stage, tumour grade and histologic tumour necrosis (all P<0.05). Furthermore, in multivariable analysis, a high plasma fibrinogen level was statistically significantly associated with a poor outcome for patients' CSS (hazard ratio (HR): 2.47, 95% confidence interval (CI): 1.49-4.11, P<0.001), MFS (HR: 2.15, 95% CI: 1.44-3.22, P<0.001) and OS (HR: 2.48, 95% CI: 1.80-3.40, P<0.001). CONCLUSION: A high plasma fibrinogen level seems to represent a strong and independent negative prognostic factor regarding CSS, MFS and OS in non-metastatic RCC patients. Thus, this easily determinable laboratory value should be considered as an additional prognostic factor for RCC patients' individual risk assessment.

Validation of the pre-treatment neutrophil-lymphocyte ratio as a prognostic factor in a large European cohort of renal cell carcinoma patients.

BACKGROUND: The neutrophil-lymphocyte ratio (NLR) has been proposed as an indicator of systemic inflammatory response. Several studies suggest a negative impact of increased NLR for patient's survival in different types of cancer. However, previous findings from small-scale studies revealed conflicting results about its prognostic significance with regard to different clinical end points in non-metastatic renal cell carcinoma (RCC) patients. Therefore, the aim of our study was the validation of the prognostic significance of NLR in a large cohort of RCC patients. METHODS: Data from 678 consecutive non-metastatic clear cell RCC patients, operated between 2000 and 2010 at a single centre, were evaluated retrospectively. Cancer-specific, metastasis-free, as well as overall survival (OS) were assessed using the Kaplan-Meier method. To evaluate the independent prognostic significance of NLR, multivariate Cox regression models were applied for all three different end points. Influence of the NLR on the predictive accuracy of the Leibovich prognosis score was determined by Harrell's concordance index. RESULTS: Multivariate analysis identified increased NLR as an independent prognostic factor for overall (hazard ratio (HR)=1.59, 95% confidence interval (CI)=1.10-2.31, P=0.014), but not for cancer-specific (HR=1.59, 95% CI=0.84-2.99, P=0.148), nor for metastasis-free survival (HR=1.39, 95% CI=0.85-2.28, P=0.184). The estimated concordance index was 0.79 using the Leibovich risk score and 0.81 when NLR was added. CONCLUSION: Regarding patients' OS, an increased NLR represented an independent risk factor, which might reflect a higher risk for severe cardiovascular and other comorbidities. Adding the NLR to well-established prognostic models such as the Leibovich prognosis score might improve their predictive ability.

Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing.

Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades ("persisting fetal microchimerism"). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles ("routine sensitivity assay") none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles ("high sensibility assay"), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril.

Evidence of fetal microchimerism in Hashimoto's thyroiditis.

Fetal microchimerism, the engraftment of fetal progenitor cells into maternal tissues, has been implicated in the etiology of autoimmune diseases. We used PCR analysis to determine whether microchimerism occurred in the thyroid glands of female patients suffering from Hashimoto's disease and thus may be involved in its etiology. PCR amplification was performed from thyroid gland specimens using primers unique to a Y-chromosomal sequence (SRY gene) and primers for a sequence that is Y/X-chromosomal homologous except for a 6-bp deletion in the X-chromosomal sequence (amelogenin). Microchimerism was detected in 8 of 17 Hashimoto patients, but in only 1 of 25 controls (nodular goiters). Both groups were of similar age and had comparable numbers of pregnancies and numbers of sons. All individuals with microchimerism had given birth to at least 1 son. Our results show that microchimerism is significantly more common in Hashimoto patients than in patients suffering from nodular goiter. We therefore suggest that microchimerism might play a role in the development of Hashimoto's disease, although we cannot completely eliminate the hypothesis that microchimerism is just an "innocent bystander" in a process triggered by other mechanisms.

CK7 expression in carcinomas of the Waldeyer's ring area.

Primary carcinomas of the Waldeyer's ring area are typically nonkeratinizing squamous cell carcinomas (SCC). Their cervical lymph node metastases are not uncommonly cystic and filled with necrotic tumor cells. Some cysts, however, contain clear fluid. During the investigation of SCC producing "fluid-filled" cystic metastases, we evaluated hematoxylin and eosin (H&E) sections of 90 primary SCC for their site of origin. We analyzed the cytokeratin (CK) profile of primary and metastatic carcinoma with special focus on the expression of CK7, a putative marker for ductal differentiation. CK7 was expressed in submucosal minor salivary gland acini and ducts, but not in the squamous surface epithelium of the Waldeyer's ring. CK7 was expressed in 11 primary SCC (8 base of tongue/3 palatine tonsil). The CK7-positive SCC were deep-seated, arose from large excretory ducts of submucosal minor salivary glands, and showed only insignificant surface involvement. They were characterized by a solid infiltrative growth pattern of basaloid cells with focal ductal differentiation. Salivary ducts adjacent to the carcinoma showed extensive intraductal hyperplasia and metaplasia. All CK7-positive carcinomas produced CK7-positive cystic nodal metastases, most of which contained paucicellular fluid. No solid CK7-positive nodal metastases were identified. In summary, a subset of carcinomas occurring in the Waldeyer's ring area appear to arise from large excretory ducts of submucosal minor salivary glands with only limited surface involvement, express CK7, and produce CK7-positive cystic "fluid-filled" nodal metastases. The histomorphology and immunophenotype suggest that these carcinomas represent basaloid SCC arising from excretory ducts of the submucosal minor salivary glands.

Trichoblastic carcinoma ("malignant trichoblastoma") with lymphatic and hematogenous metastases.

We report an aggressively behaving malignant trichogenic tumor arising in a trichoblastoma (TB) with widespread lymphatic and hematogenous metastases in a 55-year-old man with a concomitant B-cell chronic lymphocytic leukemia. The primary tumor had been present and unchanged for as long as 40 years before excision. Typical trichogenic TB with dystrophic calcification and even ossification was still present peripheral to the malignant transformation. The malignant neoplasm consisted of basaloid cells, spindle cells arranged in fascicles and densely packed rounded nests or "cell balls." The metastases consisted of immature basaloid cells and cell balls, and the recurrences became successively more undifferentiated. The residual TB reacted with antibodies to cytokeratin (CK) 6, 8, 14, and 17 and focally to S-100; the malignant primary tumor reacted uniformly with antibodies to vimentin and only focally with antibodies to CK and S-100. The metastatic tumor had lost epidermal CK expression but maintained expression of S-100 in paraffin-embedded tissues. Trichoblastic differentiation was confirmed in frozen tissues with antibodies to hair keratins. No expression of p53 or bcl-2 was identified, but p-glycoprotein (MDR-1 gene related) was expressed by primary and metastatic tumor cells. We believe that this neoplasm is best classified as a trichoblastic carcinoma arising in a TB in association with a B-cell chronic lymphocytic leukemia. This case illustrates that TBs have the potential for malignant transformation and aggressive behavior.

Cystic lymph node metastases of squamous cell carcinoma of Waldeyer's ring origin.

We analysed in a retrospective study the frequency of cystic lymph node (LN) metastases in neck dissection specimens of 123 patients with primary squamous cell carcinoma (SCC) arising in the palatine tonsils (62 M/14 F), the base of the tongue (38 M/5 F) and the nasopharynx (2 M/2 F). Eighty-two per cent of patients had metastases (64 tonsillar SCC, 33 base of tongue SCC and all four nasopharynx SCC) in 368 LN of a total 2298 sampled LN. Thirty-nine per cent of patients had exclusively solid metastases and 37% of patients had exclusively cystic metastases. A total of 62 patients had some signs of cyst formation in one or more metastatically affected LN (27 with only histological evidence of cyst formation with luminal diameters < 5 mm, 35 with clinically detectable cyst with luminal diameter > 5 mm). Cystic metastases were more common in patients with SCC of the base of the tongue (P = 0.005), while solitary clinically evident cystic metastasis with lumina > 5 mm were found exclusively in tonsillar carcinoma (P = 0.024). In comparison with solid metastases, cyst formation was associated with N-categories (N2b and N3, P = 0.005) in SCC of the base of the tongue origin. No such association was observed for tonsillar SCC (P = 0.65). The primary mechanism of cyst formation was cystic degeneration.

Multivariate karyometric approach in differential diagnosis of follicular thyroid neoplasms. A study of 31 cases.

A retrospective analysis of 19 follicular adenomas, 12 minimally invasive follicular carcinomas and 3 widely invasive follicular carcinomas of the thyroid was performed on 5-microm-thick Feulgen-stained paraffin sections by means of a semiautomatic system for picture analysis. The major aim was to assess the potential of multiparameter karyometry for separation of the first two tumour types. Sixteen planimetric and densitometric features were defined in each case on 200-300 randomly selected nuclei and processed by a number of uni- and multivariate statistical methods. Despite predominantly significant ANOVA results a substantial overlap between tumour groups limited the practical usefulness of any karyometric feature alone. Factor and cluster analyses indicated independence of planimetric and densitometric parameters from each other, which was of crucial importance in finding an optimal subset of variables for discriminant analysis. The classification rule derived from the latter procedure was checked by the "jack-knife" method, by classification of 3 widely invasive cancers and by hierarchical tumour clustering. Sensitivity and specificity of the model for detection of malignancy were 100% and 94.7%, respectively. A multivariate karyometric approach, when applied correctly, can be a useful tool for differentiation between follicular adenomas and minimally invasive follicular carcinomas of the thyroid.

ImmunoMax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens.

BACKGROUND: Since the introduction of mAb, immunohistochemistry has become an important tool in research and in surgical pathology. The most widely used fixative in routine histopathology is formaldehyde, and it has become the gold standard for morphologic tissue preservation. Although the molecular mechanism underlying the tissue fixation is not well understood, it has become clear that available immunoreactive Ag are progressively lost during the fixation process. For a long time, it was thought that formalin-sensitive Ag might be irreversibly destroyed during the fixation process. Although monoclonal anti-Ig Ab frequently worked inadequately, polyclonal anti-Ig Ab were shown to produce reproducible staining results. It thus appeared possible that most cellular Ag might not be irreversibly destroyed but only masked. EXPERIMENTAL DESIGN: Although some Ag may be retrieved under appropriate conditions, there might still be many for which available antigenic epitopes are still too sparse to be visualized, as observed for a large number of leukocyte differentiation Ag. One reliable approach to resolve this dilemma is the use of a combination of an optimized Ag retrieval system and a powerful immunohistochemical staining protocol introducing a biotin amplification step, in which signal amplification is accomplished by covalent deposition of biotin molecules. RESULTS: Cryostat and paraffin sections were stained with the avidin-biotin complex technique and, for comparison, with the new maximized immunohistochemical staining protocol, termed the ImmunoMax method. Each step was monitored to establish how effectively it enhanced the overall sensitivity. Although pretreatment with detergent, protease, a chaotropic substance, or microwave heating resulted in only moderately improved immunostaining, the biotinylated tyramine enhancement step proved to be the most efficient one, although the latter is not sufficient for many Ag when used without pretreatment steps. The combination of an Ag retrieval step with the biotinylated tyramine enhancement step resulted in a 100 to 10,000-fold boost in sensitivity without loss of specificity. CONCLUSIONS: With the ImmunoMax method, defined Ag can be reproducibly detected in formalin-fixed, paraffin-embedded tissues, and the sensitivity of the method is tremendously enhanced. Moreover, it also allows many previously unreactive or unsatisfactorily reactive Ag to be detected, as shown here for IgD, IgM, and CD7 with the use of mAb.