Basidiomycota strains as whole-cell biocatalysts for the synthesis of high-value natural benzaldehydes.

Substituted benzaldehydes are the most commonly used natural-occurring flavours in the world. The consumer's preference for 'natural or organic' aromas has increased the request for flavours possessing the 'natural' status. The resulting shortage of aromatic aldehydes of extractive origin, such as vanillin, veratraldehyde and piperonal, can be offset by developing a new biotechnological synthesis method. Here, we report a study on the microbiological reduction of five natural benzoic acid derivatives, namely p-anisic, vanillic, veratric, piperonylic and eudesmic acids, to produce the corresponding fragrant aldehydes. We found that different Basidiomycota strains can efficiently perform this transformation, with good chemical selectivity and tolerance to the toxicity of substrates and products. Besides confirming the carboxylic acid reductase activity of the already studied fungi Pycnoporus cinnabarinus, we discovered that other species such as Pleurotus eryngii, Pleurotus sapidus and Laetiporus sulphureus as well as the non-ligninolytic fungi Lepista nuda are valuable microorganisms for the synthesis of anisaldehyde, vanillin, veratraldehyde, piperonal and 3,4,5-trimethoxybenzaldehyde from the corresponding acids. According to our findings, we propose a reliable process for the preparation of the above-mentioned aldehydes, in natural form. KEY POINTS: • Fragrant benzaldehydes were obtained by biotransformation. • Basidiomycota strains reduced substituted benzoic acid to the corresponding aldehydes. • Anisaldehyde, vanillin, veratraldehyde, piperonal and 3,4,5-trimethoxybenzaldehyde were prepared in natural form.

No Evidence Was Found for the Presence of Terreolides, Terreumols or Saponaceolides H-S in the Fruiting Bodies of Tricholoma terreum (Basidiomycota, Agaricales).

Two different collections of the gilled wild fungus Tricholoma terreum, collected in Italy, were subjected to phytochemical analysis. The fungal material was confidently identified by analysis of the ITS genomic sequences. Using both HR-LC-MS and NMR techniques, no evidence was found for the presence in the fruiting bodies of terreolides, terreumols or saponaceolides H-S, in striking contrast with the isolation of these terpenoids by Chinese authors from a mushroom collected in France and identified as T. terreum. The main cytotoxic terpenoid identified and isolated from the extracts of the specimens investigated in this work was the C30 derivative saponaceolide B, which had been previously isolated from T. saponaceum and other T. terreum collections. Although saponaceolide B is a rather labile molecule, easily degradable by heat or in acidic conditions, our study indicated that none of the extraction protocols used produced saponaceolide H-S or terreolide/terreumol derivatives, thus excluding the possibility that the latter compounds could be extraction artifacts. Considered together, these findings point to the need for the unambiguous identification of mushroom species belonging to the complex genus Tricholoma, characterized by high variability in the composition of metabolites. Moreover, based on our data, T. terreum must be considered an edible mushroom.

From Waste to Value: Recent Insights into Producing Vanillin from Lignin.

Vanillin, one of the most widely used and appreciated flavoring agents worldwide, is the main constituent of vanilla bean extract, obtained from the seed pods of various members belonging to the Orchidaceae family. Due to the great demand in the food confectionery industry, as well as in the perfume industry, medicine, and more, the majority of vanillin used today is produced synthetically, and only less than one percent of the world's vanilla flavoring market comes directly from the traditional natural sources. The increasing global demand for vanillin requires alternative and overall sustainable new production methods, and the recovery from biobased polymers, like lignin, is an environmentally friendly alternative to chemical synthesis. The present review provides firstly an overview of the different types of vanillin, followed by a description of the main differences between natural and synthetic vanillin, their preparation, the market of interest, and the authentication issues and the related analytical techniques. Then, the review explores the real potentialities of lignin for vanillin production, presenting firstly the well-assessed classical methods and moving towards the most recent promising approaches through chemical, biotechnological and photocatalytic methodologies, together with the challenges and the principal issues associated with each technique.

Distinguishing Gastric/Esophageal Adenocarcinoma from Pancreatic Adenocarcinoma Using Methylation-Based Droplet Digital PCR.

The goal of this study was to develop a methylation-based droplet digital PCR to separate 2 cancer classes that do not have sensitive and specific immunohistochemical stains: gastric/esophageal and pancreatic adenocarcinomas. The assay used methylation-independent primers and methylation-dependent probes to assess a single differentially methylated CpG site; analyses of array data from The Cancer Genome Atlas network showed that high methylation at the cg06118999 probe supports the presence of cells originating from the stomach or esophagus (eg, as in gastric metastasis), whereas low methylation suggests that these cells are rare to absent (eg, pancreatic metastasis). On validation using formalin-fixed paraffin-embedded primary and metastatic samples from our institution, methylation-based droplet digital PCR targeting the corresponding CpG dinucleotide generated evaluable data for 60 of the 62 samples (97%) and correctly classified 50 of the 60 evaluable cases (83.3%), mostly adenocarcinomas from the stomach or pancreas. This ddPCR was created to be easy-to-interpret, rapid, inexpensive, and compatible with existing platforms at many clinical laboratories. We suggest that similarly accessible PCRs could be developed for other differentials in pathology that do not have sensitive and specific immunohistochemical stains.

Peptide-Mediated Targeted Delivery of Aloe-Emodin as Anticancer Drug.

Breast cancer is one of the most diffuse cancers in the world and despite the availability of the different drugs employed against it, the need for new and particularly more specific molecules is ever growing. In this framework, natural products are increasingly assuming an important role as new anticancer drugs. Aloe-emodin (AE) is one of the best characterized molecules in this field. The functionalization of bioactive natural products with selected peptide sequences to enhance their bioavailability and specificity of action is a powerful and promising strategy. In this study, we analyzed the cell specificity, cell viability effects, intracellular distribution, and immune cell response of a new peptide conjugate of Aloe-emodin in SKBR3 and A549 cell lines by means of viability tests, flow cytometry, and confocal microscopy. The conjugate proved to be more effective at reducing cell viability than AE in both cell lines. Furthermore, the results showed that it was mainly internalized within the SKBR3 cells, showing a nuclear localization, while A459 cells displayed mainly a cytoplasmic distribution. A preserving effect of the conjugate on NKs' cell function was also observed. The designed conjugate showed a promising specific activity towards HER2-expressing cells coupled with an enhanced water solubility and a higher cytotoxicity; thus, the resulting proof-of-concept molecule can be further improved as an anticancer compound.

Fractionation of Raw and Parboiled Rice Husks with Deep Eutectic Solvents and Characterization of the Extracted Lignins towards a Circular Economy Perspective.

In the present work, rice husks (RHs), which, worldwide, represent one of the most abundant agricultural wastes in terms of their quantity, have been treated and fractionated in order to allow for their complete valorization. RHs coming from the raw and parboiled rice production have been submitted at first to a hydrothermal pretreatment followed by a deep eutectic solvent fractionation, allowing for the separation of the different components by means of an environmentally friendly process. The lignins obtained from raw and parboiled RHs have been thoroughly characterized and showed similar physico-chemical characteristics, indicating that the parboiling process does not introduce obvious lignin alterations. In addition, a preliminary evaluation of the potentiality of such lignin fractions as precursors of cement water reducers has provided encouraging results. A fermentation-based optional preprocess has also been investigated. However, both raw and parboiled RHs demonstrated a poor performance as a microbiological growth substrate, even in submerged fermentation using cellulose-degrading fungi. The described methodology appears to be a promising strategy for the valorization of these important waste biomasses coming from the rice industry towards a circular economy perspective.

Combined mental task and metaboreflex impair cerebral oxygenation in patients with type 2 diabetes mellitus.

Cardiovascular regulation is altered by type 2 diabetes mellitus (DM2), producing an abnormal response to muscle metaboreflex. During physical exercise, cerebral blood flow is impaired in patients with DM2, and this phenomenon may reduce cerebral oxygenation (COX). We hypothesized that the simultaneous execution of a mental task (MT) and metaboreflex activation would reduce COX in patients with DM2. Thirteen individuals suffering from DM2 (6 women) and 13 normal age-matched controls (CTL, 6 women) participated in this study. They underwent five different tests, each lasting 12 min: postexercise muscle ischemia (PEMI) to activate the metaboreflex, control exercise recovery (CER), PEMI + MT, CER + MT, and MT alone. COX was evaluated using near-infrared spectroscopy with sensors applied to the forehead. Central hemodynamics was assessed using impedance cardiography. We found that when MT was superimposed on the PEMI-induced metaboreflex, patients with DM2 could not increase COX to the same extent reached by the CTL group (101.13% ± 1.08% vs. 104.23% ± 2.51%, P < 0.05). Moreover, patients with DM2 had higher mean blood pressure and systemic vascular resistance as well as lower stroke volume and cardiac output levels compared with the CTL group, throughout our experiments. It was concluded that patients with DM2 had reduced capacity to enhance COX when undertaking an MT during metaboreflex. Results also confirm that patients with DM2 had dysregulated hemodynamics during metaboreflex, with exaggerated blood pressure response and vasoconstriction. This may have implications for these patients' lack of inclination to exercise.

Unexpected histopathological findings after sleeve gastrectomy.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) represents one of the most commonly performed bariatric procedures and, in contrast to the Roux-en-Y gastric bypass, produces a specimen for pathologic examination. This study aims to describe unexpected histopathological findings in order to better define preoperative management of patients undergoing LSG. METHODS: All LSG cases performed at an academic center in Toronto, Ontario between 2010 and 2017 were reviewed. All specimens underwent histopathological assessment, while those with findings suspicious for neoplasia or the presence of Helicobacter pylori underwent additional immunohistochemical stainings. Baseline patient characteristics and surgical outcomes were obtained from our internal database. RESULTS: A total of 222 patients underwent LSG during the study period and had their specimens examined histologically. Among them, 22.5% underwent preoperative endoscopy. The most common histopathological diagnosis was no abnormal findings (50.9%) followed by gastritis (25.7%). Abnormal findings warranting a change in postoperative management or follow-up were discovered in 8.6% of specimens and included H. pylori infection, intestinal metaplasia, malignancy, and atrophic gastritis. Only 4.7% of all patients had not undergone preoperative endoscopy and had truly unexpected findings. No significant association was found between abnormal findings and age, sex, or baseline body mass index (BMI). CONCLUSIONS: Although a majority of patients had a gastric specimen within normal limits, 8.6% had findings requiring a change in postoperative management. This rate dropped to 4.7% when patients whose diagnoses were known preoperatively were excluded. Our findings suggest that further research is needed to better define the role of preoperative endoscopy to potentially reduce the number of unexpected findings following LSG.

Enzymes, Biocatalysis and Chemical Biology.

Chemical transformations that take advantage of biocatalysis are of great interest to chemists [...].

Bacterial Biotransformation of Oleic Acid: New Findings on the Formation of γ-Dodecalactone and 10-Ketostearic Acid in the Culture of Micrococcus luteus.

Microbial conversion of oleic acid (1) to form value-added industrial products has gained increasing scientific and economic interest. So far, the production of natural lactones with flavor and fragrance properties from fatty acids by non-genetically modified organisms (non-GMO) involves whole cells of bacteria catalyzing the hydration of unsaturated fatty acids as well as yeast strains responsible for further ß-oxidation processes. Development of a non-GMO process, involving a sole strain possessing both enzymatic activities, significantly lowers the costs of the process and constitutes a better method from the customers' point of view regarding biosafety issues. Twenty bacteria from the genus of Bacillus, Comamonas, Dietzia, Gordonia, Micrococcus, Pseudomonas, Rhodococcus and Streptomyces were screened for oxidative functionalization of oleic acid (1). Micrococcus luteus PCM525 was selected as the sole strain catalyzing the one-pot transformation of oleic acid (1) into natural valuable peach and strawberry-flavored γ-dodecalactone (6) used in the food, beverage, cosmetics and pharmaceutical industries. Based on the identified products formed during the process of biotransformation, we clearly established a pathway showing that oleic acid (1) is hydrated to 10-hydroxystearic acid (2), then oxidized to 10-ketostearic acid (3), giving 4-ketolauric acid (4) after three cycles of ß-oxidation, which is subsequently reduced and cyclized to γ-dodecalactone (6) (Scheme 1). Moreover, three other strains (Rhodococcus erythropolis DSM44534, Rhodococcus ruber PCM2166, Dietzia sp. DSM44016), with high concomitant activities of oleate hydratase and alcohol dehydrogenase, were identified as efficient producers of 10-ketostearic acid (3), which can be used in lubricant and detergent formulations. Considering the prevalence of γ-dodecalactone (6) and 10-ketostearic acid (3) applications and the economic benefits of sustainable management, microbial bioconversion of oleic acid (1) is an undeniably attractive approach.

E-cadherin can limit the transforming properties of activating ß-catenin mutations.

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in ß-catenin (CTNNB1). We have compared the dynamics and the potency of ß-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of ß-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of ß-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of ß-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:ß-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of ß-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of ß-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated ß-catenin.

Colorectal large-cell neuroendocrine carcinoma with lymphoid stroma: further evidence confirming a unique subtype associated with MLH1/PMS2 loss, BRAF mutation, Epstein-Barr virus negativity, and the possibility of a better prognosis.

AIMS: Pure large-cell neuroendocrine carcinoma (LCNEC) is an uncommon but well-recognised primary tumour of the colorectum. It is characterised by large cells organised in a discernible neuroendocrine pattern with >20 mitoses per 10 high-power fields (HPFs), and/or a Ki67 index of >20%, and coagulative tumour necrosis is invariably present. These features are supplemented by positivity for neuroendocrine immunohistochemical markers. The aim of this study was to highlight three primary LCNECs of the colorectum. METHODS AND RESULTS: All three patients were elderly females, aged 79, 85 and 89 years, who presented with ascending colon (two) tumours and a rectal tumour. All three tumours were large, ulcerated, polypoid masses (85, 76 and 55 mm; average size 72 mm) and were pT3N2. Histologically, the tumours were composed of packets and nests of cells with rosettes; other areas were in sheets composed of large cells. The mitotic counts were 23/10 HPFs, 27/10 HPFs and 24/10 HPFs, respectively. There was no mucosal dysplasia, precursor adenoma or associated adenocarcinoma component. All cases contained mainly peritumoral lymphoid aggregates, with smaller numbers of intratumoral lymphocytes. Synaptophysin, chromogranin (less intensely) and epithelial markers were expressed in all cases in the majority of the tumour cells. The Ki67 proliferative indices were 95%, 100%, and 80%, respectively. Two patients survived for 48 and 72 months, whereas the third is alive after 12 months without evidence of recurrence. CONCLUSIONS: These unusual primary colorectal LCNECs with an accompanying lymphoid component are characterised by loss of MLH1/PMS2, BRAF mutation, microsatellite instability, and Epstein-Barr virus negativity. The patients also have better overall survival than those with LCNECs lacking an accompanying lymphoid component.

Management and surveillance of non-functional pancreatic neuroendocrine tumours: Retrospective review.

BACKGROUND: /Objective. To determine the outcomes of a non-operative management approach for sporadic, small, non-functional pancreatic neuroendocrine tumours. METHODS: A retrospective chart review of patients with non-functional pancreatic neuroendocrine tumours initially managed non-operatively at a single institution was performed. Patients were identified through a search of radiologic reports, and individuals with ≥2 cross-sectional imaging studies performed >6 months apart from Jan. 1, 2000 to Dec. 31, 2013 were included. Data on tumour size, radiologic characteristics at diagnosis, interval radiologic growth, and surgical outcomes were recorded. RESULTS: Over the thirteen-year study period, 95 patients met inclusion criteria and were followed radiologically for a median of 36 months (18-69 months). Median initial tumour size on first imaging was 14.0 mm (IQR 10-19 mm). Median overall tumour growth rate was 0.03 mm/month (IQR: 0.00-0.14 mm/month). There was no significant relationship between initial tumour size and growth rate for tumours ≤ 2 cm or for lesions between 2 and 4 cm. Thirteen (14%) patients initially managed non-operatively underwent resection during the follow-up period. Reasons for surgery included interval tumour growth, patient anxiety or preference, or diagnostic uncertainty. Median time to surgery was 14 months (IQR 8-19 months). No patients progressed beyond resectability or developed metastatic disease during the observation period. CONCLUSION: For patients with sporadic, small, non-functional pancreatic neuroendocrine tumours, radiologic surveillance appears to be a safe initial approach to management.

A General Strategy for the Stereoselective Synthesis of the Furanosesquiterpenes Structurally Related to Pallescensins 1⁻2.

Here, we describe a general stereoselective synthesis of the marine furanosesquiterpenes structurally related to pallescensins 1-2. The stereoisomeric forms of the pallescensin 1, pallescensin 2, and dihydropallescensin 2 were obtained in high chemical and isomeric purity, whereas isomicrocionin-3 was synthesized for the first time. The sesquiterpene framework was built up by means of the coupling of the C10 cyclogeranyl moiety with the C5 3-(methylene)furan moiety. The key steps of our synthetic procedure are the stereoselective synthesis of four cyclogeraniol isomers, their conversion into the corresponding cyclogeranylsulfonylbenzene derivatives, their alkylation with 3-(chloromethyl)furan, and the final reductive cleavage of the phenylsulfonyl functional group to afford the whole sesquiterpene framework. The enantioselective synthesis of the α-, 3,4-dehydro-γ- and γ-cyclogeraniol isomers was performed using both a lipase-mediated resolution procedure and different regioselective chemical transformations.

Fungi-Mediated Biotransformation of the Isomeric Forms of the Apocarotenoids Ionone, Damascone and Theaspirane.

In this work, we describe a study on the biotransformation of seven natural occurring apocarotenoids by means of eleven selected fungal species. The substrates, namely ionone (α-, ß- and γ-isomers), 3,4-dehydroionone, damascone (α- and ß-isomers) and theaspirane are relevant flavour and fragrances components. We found that most of the investigated biotransformation reactions afforded oxidized products such as hydroxy- keto- or epoxy-derivatives. On the contrary, the reduction of the keto groups or the reduction of the double bond functional groups were observed only for few substrates, where the reduced products are however formed in minor amount. When starting apocarotenoids are isomers of the same chemical compound (e.g., ionone isomers) their biotransformation can give products very different from each other, depending both on the starting substrate and on the fungal species used. Since the majority of the starting apocarotenoids are often available in natural form and the described products are natural compounds, identified in flavours or fragrances, our biotransformation procedures can be regarded as prospective processes for the preparation of high value olfactory active compounds.

Mucin-rich variant of traditional serrated adenoma: a distinct morphological variant.

AIMS: Traditional serrated adenomas (TSAs) account for 5% of serrated polyps, and have a villiform architecture, eosinophilic cells with a brush border, and indented, flat-topped luminal serrations. However, some are composed of mucin-filled goblet cells (GCs): mucin-rich TSA (MrTSA). The aim of this study was to determine whether this variant has unique features as compared with classic TSA (cTSA). METHODS AND RESULTS: One hundred and fifty-six TSAs were retrieved from the period 2010-2016. Patient demographics, site of polyps and 16 microscopic variables were evaluated. TSAs containing ≥50% GCs were classified as MrTSAs. Ectopic crypt foci (ECFs) were quantified as low (1-10) or high (>10), counted at ×200 magnification, and the average was taken for 10 fields. Twenty-four fulfilled the criteria for MrTSA. In males, MrTSAs (65%) were more prevalent than cTSAs (55%). There was no age difference, and both variants had a predilection for the left colon, although, in the right colon, MrTSAs were more frequent (39%) than cTSAs (10%) (P = 0.012). Adenomatous dysplasia was present in four of 24 MrTSAs (low grade, 3; high grade, 1). The most distinctive features of MrTSAs were: a variable growth pattern [endophytic (9%), mixed (30%), or villiform/exophytic (61%)], and a lower frequency of ECFs (P = 0.001) and more intraepithelial lymphocytes (P < 0.05) than in cTSAs. MrTSAs retain characteristic luminal serrations, at least focally. Inflamed MrTSAs can mimic inflammatory polyps and hamartomatous polyps (when there are >95% GCs). CONCLUSIONS: MrTSA is characterized by >50% GCs, and fewer ECFs than cTSA, but with preservation of archetypal luminal serrations. Awareness of this variant will prevent misdiagnosis, given the association of TSA with the accelerated pathway to colorectal cancer.

Colonic mucosubmucosal elongated polyp: report of a series of 14 cases and review of the literature.

AIMS: Most colorectal polyps can be reliably assigned to one of the known polyp categories, but a subset of polyps named colonic mucosubmucosal elongated polyps (CMSEPs) do not fall into any of these categories. First described in the Japanese literature, CMSEPs seem to be under-recognized in the Western literature. The aims of this study were to describe the clinicopathological features of 14 CMSEPs, discuss potential pathogenetic mechanisms, and increase awareness of this entity among pathologists. METHODS AND RESULTS: Fourteen pedunculated colorectal polyps that met the histopathological criteria for CMSEP (as described by Matake et al. and Alizart et al.) were assessed (12 males and two females; mean age 59.7 years). Five polyps were located in the sigmoid colon, four in the rectum, two in the descending colon, and three in the colon not otherwise specified. Nine of 14 polyps were discovered incidentally: two of nine on routine screening colonoscopy, two of nine on surveillance colonoscopy for inflammatory bowel disease (IBD), and five of nine upon surgical intervention for carcinoma or IBD. None coexisted with diverticular disease. The polyps were long and slender, varied from 5 to 30 mm in length (mean 15.9 mm), and showed a normal-looking colonic mucosal layer and underlying loose submucosa with thick-walled and congested blood vessels and lymphatics. CONCLUSIONS: CMSEPs show subtle but distinctive pathological features, and occur in normal and diseased colons. Pathologists need to be aware of this entity, to avoid confusion with other more commonly encountered colorectal polyps. With increasing colon cancer screening programmes and surveillance colonoscopy, it is likely that CMSEPs will be encountered more often.

Histological overlap between colorectal villous/tubulovillous and traditional serrated adenomas.

AIMS: To ascertain the degree of histological overlap between conventional villous/tubulovillous (VA/TVAs) and traditional serrated adenomas (TSA). METHODS AND RESULTS: A total of 180 polyps from the left colon/rectum diagnosed as VA/TVAs were retrieved randomly and reviewed by five pathologists looking specifically at luminal serration, cytoplasmic eosinophilia and the presence of ectopic crypt foci (ECF). For comparative purposes, 100 tubular adenomas and 80 TSAs were also examined. Twenty VA/TVAs were reclassified as TSA. Luminal serration as noted in TSA was not seen in any of the remaining 160 polyps, ECFs were noted in 55 of the 160 VA/TVAs (34%), while cytoplasmic eosinophilia (constituting <50% of the adenoma) was noted in only 10 of 160 cases (6.2%). CONCLUSIONS: Ectopic crypt foci and cytoplasmic eosinophilia are encountered in sporadic VA/TVAs but not to the same extent and degree as in TSA. ECFs were found in one-third of cases, but cytoplasmic eosinophilia is rare. The pattern of luminal serration in TSA is very characteristic and not recapitulated in VA/TVA. The occurrence of all three histological features together occurs only in TSA. ECFs are not a sine qua non for TSA and are encountered commonly in VA/TVAs. VA/TVAs often contain occasional glands typical of TSA.

Recent Advances in the Synthesis of Carotenoid-Derived Flavours and Fragrances.

Carotenoids are important isoprenoid compounds whose oxidative degradation produces a plethora of smaller derivatives, called apocarotenoids, which possess a range of different chemical structures and biological activities. Among these natural products, compounds having less than 15 carbon atoms in their frameworks are often relevant flavours or fragrances and their manufacturing represents an important economic resource for chemical companies. The strict correlation between stereochemical structure and odour has made the stereospecific synthesis of the latter biological active compounds increasingly important. In this review, the recent advances on the synthesis of the most relevant carotenoid-derived flavours and fragrances are discussed. In particular, the new synthetic methods that have given new and innovative perspectives from a scientific standpoint and the preparative approaches that might possess industrial importance are described thoroughly.

Exome sequencing identifies nonsegregating nonsense ATM and PALB2 variants in familial pancreatic cancer.

We sequenced 11 germline exomes from five families with familial pancreatic cancer (FPC). One proband had a germline nonsense variant in ATM with somatic loss of the variant allele. Another proband had a nonsense variant in PALB2 with somatic loss of the variant allele. Both variants were absent in a relative with FPC. These findings question the causal mechanisms of ATM and PALB2 in these families and highlight challenges in identifying the causes of familial cancer syndromes using exome sequencing.