Thermal Decomposition of Cocaine and Methamphetamine Investigated by Infrared Spectroscopy and Quantum Chemical Simulations.

Examination of thermal decomposition of street samples of cocaine and methamphetamine shows that typical products detected in previous studies are accompanied by a wide palette of simple volatile compounds easily detectable by spectral techniques. These molecules increase smoke toxicity and their spectral detection can be potentially used for identification of drug samples by well-controlled laboratory thermolysis in temperature progression. In our study, street samples of cocaine and methamphetamine have been thermolyzed under vacuum over the temperature range of 350-650 °C. The volatile products (CO, HCN, CH4, C2H4, etc.) have been monitored by high-resolution Fourier-transform infrared (FTIR) spectrometry in this temperature range. The decomposition mechanism has been additionally examined theoretically by quantum-chemical calculations for the highest temperature achieved experimentally in our study and beyond. Prior to analysis, the street samples have also been characterized by FTIR, Raman spectroscopy, energy-dispersive X-ray spectroscopy, and melting point determination.

Results of morphological screening for Lynch syndrome during the period 2013-2016.

The introduction of a screening system for Lynch syndrome in pathology laboratories in Plzen yielded 24 diagnoses of Lynch syndrome during the period of 2013-2016, 20 of them presenting with colorectal cancer. In 8 of those 24 cases germline mutations of MMR genes, previously not recognized as pathogenic with certainty, were detected. Although the frequency of Lynch syndrome in patients with colorectal cancer was only 0.34 % in total, following introduction of the universal immunohistochemical investigation of MMR (mismatch repair) proteins expression in all colorectal cancers examined in Sikl´s Institute of Pathology the frequency per year in this department reached 2.4 %. The results favor universal immunohistochemical screening for Lynch syndrome in colorectal and endometrial cancer cases over a selective approach based on a combination of clinical and morphological criteria. Increased effectiveness of the universal approach is not brought about only by higher sensitivity of the immunohistochemical examination per se, but also by the possibility of automation of the process leading to increased adherence even of pathologists not directly engaged in Lynch syndrome management. However, the introduction of a nation-wide universal screening system requires support from the government and health insurance companies. Keywords: colorectal cancer - endometrial cancer - immunohistochemistry - Lynch syndrome - MMR - screening.

Programmed death-1 (PD-1) receptor/PD-1 ligand (PD-L1) expression in fumarate hydratase-deficient renal cell carcinoma.

Fumarate hydratase-deficient renal cell carcinoma (FH-RCC) is a rare and aggressive tumor affecting mostly younger patients. This is the first study to assess the expression of programmed death-1 (PD-1) receptor/PD-1 ligand (PD-L1) in FH-RCC. Formalin-fixed paraffin-embedded samples from 13 FH-RCCs collected in an international multi-institutional study, were evaluated by immunohistochemistry (IHC) for PD-1/PD-L1 reactivity in tumor cells and tumor infiltrating lymphocytes (TILs). PD-1/PD-L1 expression was further evaluated by qPCR. By IHC, PD-1 was negative in tumor cells in all 13 cases. PD-L1 was positive in tumor cells in 2/13 cases, weak positive in 7/13, and negative in 4/13 cases, respectively. In TILs, PD-1 was positive in 1/13, weak positive in 3/13, and negative in 9/13 cases. In TILs, PD-L1 was weak positive by IHC in 5/13, and negative in 8/13 cases, respectively. qPCR confirmed the result for 2 of 3 IHC weak positive PD-1 samples. Of 7 IHC weak positive samples (in tumor cells), PD-L1 mRNA was detected in all 7 tumors. The majority of FH-RCCs did not express PD-1/PD-L1 by IHC, which was confirmed by molecular analysis. PD-1/PD-L1 expression in FH-RCC is restricted to a proportion of cases which may benefit from targeted therapies.

Cutaneous Sebaceous Lesions in a Patient With MUTYH-Associated Polyposis Mimicking Muir-Torre Syndrome.

A 76-year-old white male with a history of adenocarcinoma of the rectosigmoideum and multiple colonic polyps removed at the age of 38 and 39 years by an abdominoperitoneal amputation and total colectomy, respectively, presented with multiple whitish and yellowish papules on the face and a verrucous lesion on the trunk. The lesions were surgically removed during the next 3 years and a total of 13 lesions were investigated histologically. The diagnoses included 11 sebaceous adenomas, 1 low-grade sebaceous carcinoma, and 1 squamous cell carcinoma. In some sebaceous lesions, squamous metaplasia, intratumoral heterogeneity, mucinous changes, and peritumoral lymphocytes as sometimes seen in sebaceous lesions in Muir-Torre syndrome were noted. Mutation analysis of the peripheral blood revealed a germline mutation c.692G>A,p.(Arg231His) in exon 9 and c.1145G>A, p.(Gly382Asp) in exon 13 of the MUTYH gene. A KRAS mutation G12C (c.34G>T, p.Gly12Cys) was detected in 1 sebaceous adenoma and a NRAS mutation Q61K (c.181C>A, p.Gln61Lys) was found in 2 other sebaceous adenomas. No germline mutations in MLH1, MSH2, MSH6 and PMS2 genes, no microsatellite instability, no aberrant methylation of MLH1 promoter, and no somatic mutations in MSH2 and MSH6 were found. An identical MUTYH germline mutation was found in the patient's daughter. Despite striking clinicopathological similarities with Muir-Torre syndrome, the molecular biologic testing confirmed the final diagnosis of MUTYH-associated polyposis.

Aggressive Extraocular Sebaceous Carcinoma of the Scalp Involving the Brain in a Patient With Muir-Torre Syndrome.

This article reports an unusual case of aggressive extraocular sebaceous carcinoma located on the scalp with subsequent usurpation of the bone and penetrating through the bone and meninges to the brain in a 56-year-old man affected by Muir-Torre syndrome. Microscopically, the sebaceous neoplasm was located in the middle to deep dermis without any connection to the epidermis and showed a multinodular growth with neoplastic nodules with a central comedo-type necrosis separated from each other by fibrovascular stroma. The nodules were composed of varying proportions of mature sebaceous cells and atypical basaloid cells with high degree of atypia, including high nuclear/cytoplasmic ratio, nuclear pleomorphism, macronucleoli, atypical mitoses, and necrosis. The neoplasm was totally removed. Histopathological examinations of the recurrent lesion showed identical morphological features and, in addition, signs of the tumors growing through the periosteum were noted. In the final excision specimen, both the dura mater and the brain tissue were infiltrated by the sebaceous carcinoma. The diagnosis of Muir-Torre syndrome was confirmed by molecular genetic investigation that revealed an identical germline mutation in MSH2 gene in several family members, some of whom had colorectal tumors.

[Lynch syndrome in the hands of pathologists]. / Lynchuv syndrom v rukách patologa.

Lynch syndrome (formerly hereditary non-polyposis colorectal cancer) is the most common familial colorectal cancer syndrome with a known molecular genetic background. The syndrome is caused by a germline mutation of one of the genes encoding mismatch repair (MMR) proteins that are responsible for DNA replication errors repair. Impaired function of these proteins leads to microsatellite instability (MSI) and forms a suitable background for the development and progression of tumors, mainly colorectal cancer. Traditionally, Lynch syndrome was regarded to be responsible for 2 % of all cases of colorectal cancer, however recent estimates reach even 5 %. Due to this relatively high frequency, familial occurence, the absence of the premorbid phenotype and the development of malignant tumors during the productive years of life, the correct diagnosis becomes not only a medical, but also a socioeconomical problem. Unfortunately, clinical means of diagnostics of Lynch syndrome (like the Amsterdam criteria and Bethesda guidelines) lack sensitivity. It was shown that predictive models based on histological signs of MSI are more sensitive than the clinical criteria used to detect patients suspicious of Lynch syndrome. Of all MSI-H colorectal cancers, 1/5 is caused by Lynch syndrome, the rest being only sporadic cancers caused by epigenetic inactivation of a MMR protein. To rule out the sporadic cases, molecular genetic investigation of the BRAF gene and methylation analysis of MLH1 is used in the diagnostic workup of Lynch syndrome. The suspicion of Lynch syndrome, based on the results of the assortment of diagnostic methods mentioned above, should be proven by detection of a germline mutation of an MMR gene in peripheral blood, and followed by screening of family members, which is a necessary condition for efficient prevention.

Choriogonadotropin positive seminoma-a clinicopathological and molecular genetic study of 15 cases.

The presence of human chorionic gonadotropin (hCG) positive syncytiotrophoblastic cells (STC) in classic seminoma (CS) is well documented. CS with extensive hCG positive, non-syncytiotrophoblastic tumour cells (without STC) is exceptionally rare. In this study, we present 15 such cases. 168 CSs were retrieved from the Plzen Tumor registry. Cases of mixed germ cell tumors (with CS) and CSs with typical STC were excluded. Cases with completely embedded tumor mass were selected for further study and immunohistochemically examined with anti-hCG. Positive cases were further analyzed by reverse transcriptase polymerase chain reaction. Two groups of hCG-positive CSs were identified. Group 1 comprised 10 patients with a mean patient age of 37.7 years and mean tumor size of 4.96 cm. Eight cases were pT1 (TMN 2009) and 2 cases pT3a. Blood levels of hCG were elevated in 6 of the 10 patients preoperatively. In 2 patients the blood level of hCG was not tested. Mean follow-up period was 6.1 years. No metastatic behavior was noted. All tumors were extensively immunoreactive for hCG in more than 60% of tumor cells. The expression of hCG beta subunit (CGB)-mRNA in tumor tissue was documented. Group 2: Comprised 5 patients with a mean age was 34 years. Mean tumor size was 4.7 cm. Four cases were stage pT1 and 1 case was pT2. The mean follow-up period was 3.1 years. No metastatic behavior was noted. Preoperative blood levels of hCG were elevated in 1/5 of the patient. Strong hCG positivity was limited to scattered single tumor cells distributed throughout the entire tumor. Only weak expression of CGB mRNA was detected. We can conclude that immunohistochemical detection of expression of hCG in CS is not limited to syncytiotrophoblastic cells. In this study, we report two immunohistochemical patterns of hCG expression in classic seminomas: diffuse hCG staining in the majority of tumor cells and scattered hCG-positive cells within the tumor.

Adolescent Onset of Localized Papillomatosis, Lymphedema, and Multiple Beta-Papillomavirus Infection: Epidermal Nevus, Segmental Lymphedema Praecox, or Verrucosis? A Case Report and Case Series of Epidermal Nevi.

Herein, we report the case of a 12-year-old female who noted the recent onset of an oval, circumscribed, 10-cm papillomatous plaque affecting the thigh and vulva that showed histologic signs of lymphedema without evidence of secondary lymphedema. The sequencing of genes associated with a delayed onset of lymphedema or epidermal nevi (EN) - GATA2 and GJC2, and HRAS and KRAS, respectively - showed wild-type alleles. Polymerase chain reaction for human papillomavirus (HPV) DNA demonstrated infections with 15 HPV genotypes. Evidence of productive HPV infection, HPV capsid expression, and cytopathic changes was detected. At the 6-month follow-up, no evidence of recurrence was found after complete excision. The analysis of a consecutive series of 91 EN excision specimens revealed that 76% exhibited histologic evidence of lymphostasis. Notably, multiple acrochordon-like EN, which most closely resembled this case, showed similar signs of localized lymphedema. The late onset and evidence of lymphedema favors the diagnosis of congenital unisegmental lymphedema. However, the clinical findings and epidermal changes point to the diagnosis of EN. Moreover, localized verrucosis also accurately describes this patient's cutaneous findings. Based on the above evidence, we postulate that an abnormal development of lymphatics may play a primary role in the pathogenesis of some types of EN and facilitate productive HPV infection.

[Sequencing - classical method]. / Sekvenování - klasická metodika.

In this article the basic methods of reading nucleotide sequences in DNA molecules are summarized. Sanger sequencing is described most thoroughly as it is the most frequent routine method currently being utilized. The article describes in detail the principle of sequence determination through the production of fragments with a known end base using chain termination synthesis of DNA and ways of separation and detection of the fragments. Some alternative methods of sequencing are mentioned in short. Basic approaches of analyzing sequence data are explained as well as different outcomes, obstacles and challenges.

Imbalance in expression of hop (Humulus lupulus) chalcone synthase H1 and its regulators during hop stunt viroid pathogenesis.

Viroid-derived small RNAs generated during hop stunt viroid (HSVd) pathogenesis may induce the symptoms found in the hop cultivar "Admiral", including observed shifts in phenylpropanoid metabolites and changes in petiole coloration. Using quantitative RT-PCR, we examined hop lupulin gland-specific genes that have been shown to be involved in phenylpropanoid metabolism, for altered expression in response to infection with two HSVd isolates, HSVd-g and CPFVd. Most notably, the expression of a gene encoding a key enzyme for phenylpropanoid synthesis, naringenin-chalcone synthase H1 (chs_H1), decreased up to 40-fold in infected samples. In addition, a marked decrease in the expression of HlbHLH2 and an increase in the expression of HlMyb3 were observed. These two genes encode transcription factors that form a ternary complex with HlWDR1 for chs_H1 promoter activation. In a transient expression assay, a decrease in the ternary complex potential to activate the chs_H1 promoter was observed upon the decrease of HlbHLH2 expression. In addition, targeting of the chs_H1 transcript by vd-sRNAs may contribute to these expression changes. Our data show that HSVd infection causes a significant imbalance in the expression of phenylpropanoid metabolite-affecting genes via a complex mechanism, possibly involving regulatory disorders and direct targeting by vd-sRNA.

Tubulocystic renal cell carcinoma: is there a rational reason for targeted therapy using angiogenic inhibition? Analysis of seven cases.

Generally, patients with renal cell carcinoma (RCC) are viewed as potential candidates for antiangiogenic targeted therapy. Tubulocystic RCC (TCRC) is a recently described entity which may behave aggressively, and the rationale for antiangiogenic therapy in this group of renal tumors has yet to be determined. Seven TCRCs and five non-tumor tissue samples from seven patients were subjected to relative expression analysis of mRNA levels of 16 genes involved in three angiogenic signal pathways: (1) VHL/HIF, (2) RTK/mitogen-activated protein kinase (MAPK), and (3) PI3K/Akt/mTOR. Two of them, pathways (2) and (3), are often targeted by antiangiogenic agents. We also determined the mutation and methylation status of the VHL gene. Finally, the levels of vascular endothelial growth factor A (VEGFA), HIF-1α, HIF-2α proteins, and phosphorylated mTOR protein were also determined. The comparison of tumor and control samples revealed no changes of mRNA levels of the following genes: VHL, HIF-1α, HIF-2α, PTEN, Akt2, Akt3, mTOR, VEGFA, KDR, HRas, C-Jun, EGFR, and FGF2. Significantly elevated mRNA level of TP53 was found, while the mRNA levels of FLT1 and C-FOS were reduced in tumor samples. No mutations or methylation in the VHL gene were found. Changes in levels of studied proteins VEGFA, HIF-1α, HIF-2α, and increased phosphorylation of mTOR protein were not found. Three studied angiogenic pathways (VHL/HIF, RTK/MAPK, and PI3K/Akt/mTOR) seem not to be upregulated in TCRC samples, so there appears to be no rationale for a general recommendation of antiangiogenic targeted therapeutic protocols for patients with these tumors.

Biological and molecular analysis of the pathogenic variant C3 of potato spindle tuber viroid (PSTVd) evolved during adaptation to chamomile (Matricaria chamomilla).

Viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small RNAs (vsRNAs). We describe a new PSTVd variant C3, evolved through sequence adaptation to the host chamomile (Matricaria chamomilla) after biolistic inoculation with PSTVd-KF440-2, which causes extraordinary strong ('lethal') symptoms. The deletion of a single adenine A in the oligoA stretch of the pathogenicity (P) domain appears characteristic of PSTVd-C3. The pathogenicity and the vsRNA pool of PSTVd-C3 were compared to those of lethal variant PSTVd-AS1, from which PSTVd-C3 differs by five mutations located in the P domain. Both lethal viroid variants showed higher stability and lower variation in analyzed vsRNA pools than the mild PSTVd-QFA. PSTVd-C3 and -AS1 caused similar symptoms on chamomile, tomato, and Nicotiana benthamiana, and exhibited similar but species-specific distributions of selected vsRNAs as quantified using TaqMan probes. Both lethal PSTVd variants block biosynthesis of lignin in roots of cultured chamomile and tomato. Four 'expression markers' (TCP3, CIPK, VSF-1, and VPE) were selected from a tomato EST library to quantify their expression upon viroid infection; these markers were strongly downregulated in tomato leaf blades infected by PSTVd-C3- and -AS1 but not by PSTVd-QFA.

Cloning and molecular analysis of HlbZip1 and HlbZip2 transcription factors putatively involved in the regulation of the lupulin metabolome in hop (Humulus lupulus L.).

Hop (Humulus lupulus L.), the essential source of beer flavor is of interest from a medicinal perspective in view of its high content in health-beneficial terpenophenolics including prenylflavonoids. The dissection of biosynthetic pathway(s) of these compounds in lupulin glands, as well as its regulation by transcription factors (TFs), is important for efficient biotechnological manipulation of the hop metabolome. TFs of the bZIP class were preselected from the hop transcriptome using a cDNA-AFLP approach and cloned from a cDNA library based on glandular tissue-enriched hop cones. The cloned TFs HlbZIP1A and HlbZIP2 have predicted molecular masses of 27.4 and 34.2 kDa, respectively, and both are similar to the group A3 bZIP TFs according to the composition of characteristic domains. While HlbZIP1A is rather neutral (pI 6.42), HlbZIP2 is strongly basic (pI 8.51). A truncated variant of HlbZIP1 (HlbZIP1B), which is strongly basic but lacks the leucine zipper domain, has also been cloned from hop. Similar to the previously cloned HlMyb3 from hop, both bZIP TFs show a highly specific expression in lupulin glands, although low expression was observed also in other tissues including roots and immature pollen. Comparative functional analyses of HlbZip1A, HlbZip2, and subvariants of HlMyb3 were performed in a transient expression system using Nicotiana benthamiana leaf coinfiltration with Agrobacterium tumefaciens strains bearing hop TFs and selected promoters fused to the GUS reference gene. Both hop bZIP TFs and HlMyb3 mainly activated the promoters of chalcone synthase chs_H1 and the newly cloned O-methyl transferase 1 genes, while the response of the valerophenone synthase promoter to the cloned hop TFs was very low. These analyses also showed that the cloned bZIP TFs are not strictly G-box-specific. HPLC analysis of secondary metabolites in infiltrated Petunia hybrida showed that both hop bZIP TFs interfere with the accumulation and the composition of flavonol glycosides, phenolic acids, and anthocyanins, suggesting the possibility of coregulating flavonoid biosynthetic pathways in hop glandular tissue.

Antitumor effects and cytotoxicity of recombinant plant nucleases.

Recombinant plant nucleases R-TBN1 and R-HBN1 were isolated to homogeneity and examined for their antitumor effects and cytotoxicity. Although antiproliferative effects of both recombinant nucleases were not significant on the ML-2 cell culture in vitro, the nucleases were strongly cytostatic in vivo after their administration intravenously as stabilized conjugates with polyethylene glycol (PEG). Recombinant nucleases were as effective against melanoma tumors as previously studied pine pollen (PN) and mung bean nucleases and their effects were reached at about 10 times lower concentrations compared to the use of bovine seminal RNase (BS-RNase). Because the recombinant nucleases R-HBN1 and R-TBN1 share only 67.4% amino acid identity and showed only partial immunochemical cross-reactivity, their similar anticancerogenic effects can be mainly explained by their catalytical similarity. Both recombinant nucleases showed lower degree of aspermatogenesis compared to BS-RNAse and PN nuclease. Unlike BS-RNase, aspermatogenesis induced by both recombinant nucleases could not be prevented by the homologous antibody complexes. Owing to relatively low cytotoxicity on the one hand, and high efficiency at low protein levels on the other, recombinant plant nucleases R-HBN1 and R-TBN1 appear to be stable biochemical agents that can be targeted as potential antitumor cytostatics.

Circadian rhythm gene regulation in the housefly Musca domestica.

The circadian mechanism appears remarkably conserved between Drosophila and mammals, with basic underlying negative and positive feedback loops, cycling gene products, and temporally regulated nuclear transport involving a few key proteins. One of these negative regulators is PERIOD, which in Drosophila shows very similar temporal and spatial regulation to TIMELESS. Surprisingly, we observe that in the housefly, Musca domestica, PER does not cycle in Western blots of head extracts, in contrast to the TIM protein. Furthermore, immunocytochemical (ICC) localization using enzymatic staining procedures reveals that PER is not localized to the nucleus of any neurons within the brain at any circadian time, as recently observed for several nondipteran insects. However, with confocal analysis, immunofluorescence reveals a very different picture and provides an initial comparison of PER/TIM-containing cells in Musca and Drosophila, which shows some significant differences, but many similarities. Thus, even in closely related Diptera, there is considerable evolutionary flexibility in the number and spatial organization of clock cells and, indeed, in the expression patterns of clock products in these cells, although the underlying framework is similar.