Cystic lesions of the retrorectal space.

Cysts of the retrorectal space comprise a heterogeneous group of rare lesions. Most develop from embryological remnants and include tailgut cysts, dermoid cysts, rectal duplication cysts, anal canal duplication cysts, sacrococcygeal teratomas and anterior meningocoele. Tailgut cyst is the most common cyst of developmental origin, usually presenting as a multilocular cystic mass with mucoid content and lined by multiple epithelial types. Compared with tailgut cysts, rectal duplication cysts display all layers of the large bowel wall including a well-defined muscularis propria. Retrorectal cysts of non-developmental origin are far less common and represent lesions that either infrequently involve the retrorectal space or undergo extensive cystic change. This review provides an overview of the various histological types of cystic lesions of the retrorectal space, divided into cysts of developmental origin and those of non-developmental origin. A practical pathological and multidisciplinary approach to diagnosing these lesions is presented.

Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble.

Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

High incidence of inflammatory bowel disease in Northern Australia: a prospective community population-based Australian incidence study in the Mackay-Isaac-Whitsunday region.

BACKGROUND AND AIMS: To determine the incidence of inflammatory bowel disease (IBD) in the Mackay-Isaac-Whitsunday region in Northern Queensland (-21.14° S) and to allow a comparison with Southern Australian and New Zealand data (Geelong, Australia -38.14° S; Tasmania -41.43° S and -42.88° S (Launceston and Hobart) and Canterbury, New Zealand -43.46 °S). DESIGN: A prospective observational community population-based IBD study was conducted between 1 June 2017 and 31 May 2018. OUTCOME MEASURES: Primary includes the crude annual incidence rate of IBD, Crohn's disease (CD), ulcerative colitis (UC) and inflammatory bowel disease-unclassified (IBDU), while secondary includes disease phenotype and behaviour. RESULTS: Fifty-six new cases of IBD were identified. Twenty-three were CD, 30 were UC and 3 were IBDU. The crude annual incidence rate per 100 000 for IBD, CD, UC and IBDU were 32.2 (95% confidence interval (CI): 24.78-41.84), 13.23 (95% CI: 8.79-19.90), 17.25 (95% CI: 12.06-24.67) and 1.73 (95% CI: 0.56-5.35). When directly age-standardised to the World Health Organisation Standard Population Distribution, the overall CD, UC and IBDU incidence were 13.19, 17.34 and 1.85 per 100 000, with an overall age-standardised IBD incidence of 32.38. CONCLUSIONS: This is the first study to define the incidence of IBD in a Northern Australian cohort and to allow a comparison between North and Southern Australia. The IBD crude is the highest reported in Australia. Like others, we found a high and low incidence of upper gastrointestinal Crohn's disease and complicated disease at diagnosis respectively, likely reflective of the increased availability and early uptake of endoscopic procedures.

Sampling and Reporting of Inflammatory Bowel Disease.

Pathologists have an important and expanding role in the diagnosis and management of inflammatory bowel disease. This role includes the initial diagnosis of the disease, assessment of the response to treatment and the identification of short-term complications such as cytomegalovirus infection and long-term complications such as dysplasia. Furthermore, the assessment of resection specimens for complication of disease is important to determining the risk of subsequent disease or inflammation within an ileal pouch. Adequate sampling of the disease at endoscopy and from the surgical resection specimen is vital to determining the ultimate information that can be provided by the pathologist. This sampling is determined by the clinical scenario. Similarly, a standardized approach to reporting and synthesizing the histologic findings will improve patient management. This is best exemplified by the increasing interest in histologic activity indices, such as the Nancy index in ulcerative colitis, and in the standardized reporting for inflammatory bowel disease dysplasia recommended by the SCENIC international consensus.

RET gene rearrangements occur in a subset of pancreatic acinar cell carcinomas.

Pancreatic acinar cell carcinoma is relatively rare (1 to 2% of pancreatic malignancies) but may be under-recognized. In contrast to pancreatic ductal adenocarcinoma, most acinar cell carcinomas lack mutations in KRAS, DPC, CDKN2A or TP53, but appear to have a high incidence of gene rearrangements, with up to 20% reported to be driven by BRAF fusions. With the development of a new class of RET-specific tyrosine kinase inhibitors, which appear to have particularly strong activity against RET gene rearranged tumours, there is now considerable interest in identifying RET gene rearrangements across a wide range of cancers. RET rearrangements have been reported to occur at a very low incidence (<1%) in all pancreatic carcinomas. We postulated that given its unique molecular profile, RET gene rearrangements may be common in acinar cell carcinomas. We performed fluorescent in-situ hybridization (FISH) studies on a cohort of 40 acinar cell spectrum tumours comprising 36 pure acinar cell carcinomas, three pancreatoblastomas and one mixed acinar-pancreatic neuroendocrine tumour. RET gene rearrangements were identified in 3 (7.5%) cases and BRAF gene rearrangements in 5 (12.5%). All gene rearranged tumours were pure acinar cell carcinomas. Our findings indicate that amongst all pancreatic carcinomas, acinar carcinomas are highly enriched for potentially actionable gene rearrangements in RET or BRAF. FISH testing is inexpensive and readily available in the routine clinical setting and may have a role in the assessment of all acinar cell carcinomas-at this stage to recruit patients for clinical trials of new targeted therapies, but perhaps in the near future as part of routine care.

RAF1 rearrangements are common in pancreatic acinar cell carcinomas.

There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 'pure' acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.

Clinicopathological features of pyloric gland adenomas of the duodenum: a multicentre study of 57 cases.

AIMS: To determine the clinicopathological features of pyloric gland adenomas (PGA) that arise in the duodenum. METHODS AND RESULTS: Fifty-seven cases of duodenal PGA were identified and analysed from 56 patients. Clinicopathological and immunohistochemical analyses were performed. PGA tend to occur in older individuals (median age = 73.5), with a slight female predominance (25 males, 31 females). PGA arise more commonly in the proximal duodenum (68.75% in D1, 25% in D2 and 6.25% in D3) and usually present as mucosal nodules (98.2%) or plaques (1.8%), with a mean size of 14.8 mm. There is associated gastric heterotopia in 22.8% of cases. PGA showing features of high-grade dysplasia were significantly larger in size than PGA, showing only low-grade dysplasia (23.1 versus 8.7 mm; P = 0.0001) and more likely to show a tubulovillous rather than a pure tubular architecture (P = 0.025). In our series, 10 of 56 patients had intramucosal or invasive carcinoma associated with the duodenal PGA (17.9%). Three of these carcinomas showed lymph node metastasis. Following definitive treatment, local recurrence occurred in only three patients. CONCLUSIONS: Duodenal PGA tend to occur in the proximal duodenum of older individuals. Larger size and tubulovillous architecture correlates with high-grade dysplasia and associated adenocarcinoma. The low recurrence rate of these lesions would suggest that endoscopic management is appropriate, provided that the lesion can be completely resected.

Spectrum of gastrointestinal tract pathology in a multicenter cohort of 43 Cowden syndrome patients.

Most patients with Cowden syndrome have lesions in the gastrointestinal tract, characterized by multiple polyps of various histologic types in the large bowel, polyps in the upper gastrointestinal tract, and esophageal glycogenic acanthosis. However, pathologists are often unaware of the distinctive polyposis phenotype of Cowden syndrome. In this multicenter study, we report the spectrum of gastrointestinal manifestations in a series of 43 Cowden syndrome patients who had at least one endoscopy. The median age at the first endoscopy was 46 years and 58% were women. In 24 of 29 (83%) tested patients, a pathogenic germline mutation in PTEN was identified. The histology from 199 endoscopy procedures (67 upper gastrointestinal endoscopy and 132 colonoscopies) was reviewed. Hamartomatous polyps of the large bowel were the most common lesions, present in 85% of patients. Hamartomatous polyps showed varied histology, including lymphoid aggregates in 55% of patients, a lipomatous component in 52%, a ganglioneuromatous component in 52%, and a fibrous-rich component in 14%. Polyps with at least two different stromal components were found in 55% of patients. Inflammatory polyps were present in 21% of patients. Conventional adenomas and serrated polyps were identified in 48% and 62% of patients, respectively. In the upper gastrointestinal tract, the most common lesions were esophageal glycogenic acanthosis (37%), gastric hamartomatous polyps (47%), and duodenal hamartomatous polyps (20%). All patients with glycogenic acanthosis who had a colonoscopy had hamartomatous polyps of the large bowel. In five patients, the diagnosis of Cowden syndrome was established after the pathology report raised suspicion for the diagnosis. Pathologists who are aware of the characteristic admixture of lesions in Cowden syndrome can play an essential role in recommending referral to genetic counseling and gene testing. Early diagnosis of Cowden syndrome is important, as these patients and their relatives are at increased risk for developing multiple cancers.

Actinomyces in Crohn's-like appendicitis.

AIMS: Appendicitis with a Crohn's-like histological appearance generally raises concern for Crohn's disease, Yersinia infection, and interval appendectomy. Actinomyces infection is a recognised cause of chronic appendicitis that can histologically mimic Crohn's disease. METHODS AND RESULTS: We report on 20 cases of appendicitis with Crohn's-like histological features that were due to Actinomyces. Most patients presented with acute or chronic abdominal pain. Imaging studies suggested a mass in five cases. Two patients had interval appendectomy. Histological features showed Crohn's-like appendicitis in 16 cases, with moderate to marked fibrosis and granulomas in seven cases. The other four cases had less consistent histological findings. None of the patients developed Crohn's disease during the follow-up interval (median, 37 months). CONCLUSIONS: Actinomyces can be associated with Crohn's-like appendicitis with marked fibrosis, transmural inflammation, lymphoid hyperplasia, and granulomas.

Gastroenteropancreatic neuroendocrine neoplasms: selected pathology review and molecular updates.

Gastroenteropancreatic (GEP) neuroendocrine neoplasms can be broadly separated into well- and poorly differentiated categories. Tumours within each category have similarities in morphology and immunophenotype, but vary in grade, behaviour, molecular signature and responses to therapy. The aetiology of these differences is multifactorial. Site of origin, mucosal milieu and hereditary influences are some of the currently known factors. Given these differences, staging and grading systems continue to evolve, and the most recent World Health Organization classification of pancreatic neuroendocrine neoplasms reflects this by introducing a grade 3 neuroendocrine tumour category for morphologically well-differentiated tumours with an elevated Ki-67 proliferation index and/or mitotic count. This review aims to highlight current classification guidelines with discussion of unique site-specific features of selected GEP neuroendocrine neoplasms and an emphasis on practical issues related to daily reporting.

Early Barrett esophagus-related neoplasia in segments 1 cm or longer is always associated with intestinal metaplasia.

The assumption that intestinal metaplasia is a prerequisite for intraepithelial neoplasia/dysplasia and adenocarcinoma in the distal esophagus has been challenged by observations of adenocarcinoma without associated intestinal metaplasia. This study describes our experience of intestinal metaplasia in association with early Barrett neoplasia in distal esophagus and gastroesophageal junction. We reviewed the first endoscopic mucosal resection of 139 patients with biopsy-proven neoplasia. In index endoscopic mucosal resection, 110/139 (79%) cases showed intestinal metaplasia. Seven had intestinal metaplasia on prior biopsy specimens and three had intestinal metaplasia in subsequent specimens, totaling 120/139 (86%) patients showing intestinal metaplasia at some point supporting the theory of sampling error for absence of intestinal metaplasia in some cases. Those without intestinal metaplasia (13%) were enriched for higher stage disease (T1a Stolte m2 or above) supporting the assertion of obliteration of intestinal metaplasia by the advancing carcinoma. All cases of intraepithelial neoplasia and T1a Stolte m1 carcinomas had intestinal metaplasia (42/42). The average density of columnar-lined mucosa showing goblet cells was significantly less in shorter segments compared to those ≥3 cm (0.31 vs 0.51, P=0.0304). Cases where segments measured less than 1 cm were seen in a higher proportion of females and also tended to lack intestinal metaplasia. We conclude that early Barrett neoplasia is always associated with intestinal metaplasia; absence of intestinal metaplasia is attributable to sampling error or obliteration of residual intestinal metaplasia by neoplasia and those with segments less than 1 cm show atypical features for Barrett-related disease (absent intestinal metaplasia and female gender), supporting that gastroesophageal junction adenocarcinomas are heterogeneous.

Galectin-1 is associated with poor prognosis in patients with cutaneous head and neck cancer with perineural spread.

Spread of head and neck cancer along the cranial nerves is often a lethal complication of this tumour. Current treatment options include surgical resection and/or radiotherapy, but recurrence is a frequent event suggesting that our understanding of this tumour and its microenvironment is incomplete. In this study, we have analysed the nature of the perineural tumour microenvironment by immunohistochemistry with particular focus on immune cells and molecules, which might impair anti-tumour immunity. Moderate to marked lymphocyte infiltrates were present in 58.8% of the patient cohort including T cells, B cells and FoxP3-expressing T cells. While human leukocyte antigen (HLA) class I and more variably HLA class II were expressed on the tumour cells, this did not associate with patient survival or recurrence. In contrast, galectin-1 staining within lymphocyte areas of the tumour was significantly associated with a poorer patient outcome. Given the known role of galectin-1 in immune suppression, the data suggest that galectin inhibitors might improve the prognosis of patients with perineural spread of cancer.

Self-limited coeliac-like enteropathy: a series of 18 cases highlighting another coeliac disease mimic.

AIMS: To describe the clinical and pathological features of a series of patients with biopsy findings of a coeliac disease-like enteropathy in the setting of an acute illness. METHODS AND RESULTS: Eighteen cases of an abrupt-onset, self-limited illness with coeliac-like enteropathy (SLCE) were collected prospectively. Medication reaction, immune disorder, food allergy and parasitic infection were excluded. Coeliac disease was excluded by the transient nature of the illness and absence of tissue transglutaminase (TTG) elevation (nine of nine) or human leucocyte antigen (HLA)-DQ2/DQ8 haplotype (eight of nine). Clinical symptoms were recorded and histopathological findings in all gastrointestinal sites were quantified. Findings in the duodenum were compared to a coeliac disease control group. In 12 cases the clinical diagnosis was infective enteritis, probably viral in type. In six cases, a definite diagnosis was not established. Histological differences from coeliac disease included intra-epithelial neutrophil infiltration (P < 0.001), fewer intra-epithelial lymphocytes (P = 0.038) and uniform or crypt predominant intra-epithelial lymphocytosis in SCLE. One case displayed pan-gastrointestinal tract lymphocytosis. All resolved within 6 months. CONCLUSIONS: Histopathologists need to be aware that a coeliac disease-like enteropathy may occur in the setting of an acute gastrointestinal illness and resolve without sequelae.

Chief cell-predominant gastric polyps: a series of 12 cases with literature review.

AIMS: Rare gastric lesions composed of a combined proliferation of chief and oxyntic cells have been variably called adenocarcinoma of fundic gland type and oxyntic gland polyp/adenoma. Herein, we present a series of cases that show a morphological spectrum of chief and oxyntic cell proliferations. METHODS AND RESULTS: Routine and consultation cases were collated from five institutions. Information regarding site, size, endoscopic appearance, clinical history and medication use, when available, was accrued, as was the histological features and immunoprofiles. A total of 12 cases were collated. Age ranged from 39 to 81 years. All the lesions were located in the fundus; seven of eight were polypoid lesions endoscopically. Lesions were primarily solitary, averaged 4.6 mm in diameter (largest 9 mm) and comprised >50% chief cells. The predominant architectural pattern was of anastomosing and solid and clustered glands or a mixture of these patterns. Lesions were limited mainly to the mucosa, although two showed submucosal involvement. None had known metastatic disease. CONCLUSIONS: This series included lesions that were previously described as gastric adenocarcinoma of fundic gland type and oxyntic gland polyp/adenoma. They are located exclusively in the fundus and composed predominantly of chief cells with low-grade cytology and appear to show a morphological continuum.

Duodenal lymphocytosis with no or minimal enteropathy: much ado about nothing?

Duodenal lymphocytosis is a nonspecific finding that is being detected with heightened frequency. Although increased intraepithelial lymphocytosis with normal villous architecture classically corresponds to grade 1 of the Marsh classification, many other conditions have been reported to be associated with this histologic pattern. In this article, we offer a broad review of the associations of isolated increased intraepithelial lymphocytosis with celiac and nonceliac gluten sensitivity, as well as of the broadening nonceliac etiologies.

Collagenous gastritis: a morphologic and immunohistochemical study of 40 patients.

Collagenous gastritis is a rare condition defined histologically by a superficial subepithelial collagen layer. This study further characterizes the morphologic spectrum of collagenous gastritis by evaluating a multi-institutional series of 40 patients (26 female and 14 male). The median age at onset was 16 years (range 3-89 years), including 24 patients (60%) under age 18. Twelve patients (30%) had associated celiac disease, collagenous sprue, or collagenous colitis. Hematoxylin and eosin slides were reviewed in biopsies from all patients and tenascin, gastrin, eotaxin, and IgG4/IgG immunohistochemical stains were applied to a subset. The distribution of subepithelial collagen favored the body/fundus in pediatric patients and the antrum in adults. There were increased surface intraepithelial lymphocytes (>25 lymphocytes/100 epithelial cells) in five patients. Three of these patients had associated celiac and/or collagenous sprue/colitis, while the remaining two had increased duodenal lymphocytosis without specific etiology. An eosinophil-rich pattern (>30 eosinophils/high power field) was seen in 21/40 (52%) patients. Seven patients' biopsies demonstrated atrophy of the gastric corpus mucosa. Tenascin immunohistochemistry highlighted the subepithelial collagen in all 21 specimens evaluated and was a more sensitive method of collagen detection in biopsies from two patients with subtle subepithelial collagen. No increased eotaxin expression was identified in 16 specimens evaluated. One of the twenty-three biopsies tested had increased IgG4-positive cells (100/high power field) with an IgG4/IgG ratio of 55%. In summary, collagenous gastritis presents three distinct histologic patterns including a lymphocytic gastritis-like pattern, an eosinophil-rich pattern, and an atrophic pattern. Eotaxin and IgG4 were not elevated enough to implicate these pathways in the pathogenesis. Tenascin immunohistochemistry can be used as a sensitive method of collagen detection.

A clinicopathological and molecular analysis of 200 traditional serrated adenomas.

The traditional serrated adenoma is the least common colorectal serrated polyp. The clinicopathological features and molecular drivers of these polyps require further investigation. We have prospectively collected a cohort of 200 ordinary and advanced traditional serrated adenomas and performed BRAF and KRAS mutational profiling, CpG island methylator phenotype analysis, and immunohistochemistry for a panel of 7 antibodies (MLH1, ß-catenin, p53, p16, Ki67, CK7, and CK20) on all cases. The mean age of the patients was 64 years and 50% were female. Of the polyps, 71% were distal. Advanced histology (overt dysplasia or carcinoma) was present in 19% of cases. BRAF mutation was present in 67% and KRAS mutation in 22%. BRAF mutant traditional serrated adenomas were more frequently proximal (39% versus 2%; P≤0.0001), were exclusively associated with a precursor polyp (57% versus 0%; P≤0.0001), and were more frequently CpG island methylator phenotype high (60% versus 16%; P≤0.0001) than KRAS mutant traditional serrated adenomas. Advanced traditional serrated adenomas retained MLH1 expression in 97%, showed strong p53 staining in 55%, and nuclear ß-catenin staining in 40%. P16 staining was lost in the advanced areas of 55% of BRAF mutant traditional serrated adenomas compared with 10% of the advanced areas of KRAS mutant or BRAF/KRAS wild-type traditional serrated adenomas. BRAF and KRAS mutant traditional serrated adenomas are morphologically related but biologically disparate polyps with distinctive clinicopathological and molecular features. The overwhelming majority of traditional serrated adenomas retain mismatch repair enzyme function indicating a microsatellite-stable phenotype. Malignant progression occurs via TP53 mutation and Wnt pathway activation regardless of mutation status. However, CDKN2A (encoding the p16 protein) is silenced nearly exclusively in the advanced areas of the BRAF mutant traditional serrated adenomas. Thus, the BRAF mutant traditional serrated adenoma represents an important precursor of the aggressive BRAF mutant, microsatellite-stable subtype of colorectal carcinoma.

Do serrated neoplasms of the small intestine represent a distinct entity? Pathological findings and molecular alterations in a series of 13 cases.

AIMS: To characterize pathological, immunohistochemical and molecular features of small intestinal serrated neoplasms. METHODS AND RESULTS: We report 13 serrated neoplasms located predominantly in the duodenum (median age, 71 years; male to female ratio, 7:6). The serrated adenomas demonstrated prominent serration, ectopic crypt formations and cytological features reminiscent of colorectal traditional serrated adenomas. Almost half the serrated adenomas demonstrated high-grade dysplasia or were associated with an adenocarcinoma. Immunohistochemical and molecular analysis showed an intestinal (CDX2-positive) phenotype in all tumours, abnormal ß-catenin staining in three cases (23%), abnormal p53 expression in four cases (31%), focal loss of MGMT expression in one case (8%), KRAS mutation in five cases (38%) and CpG island methylator phenotype in six cases (50%). A diffuse pattern of Ki67 expression was present in eight adenomas (62%) and was associated with high-grade dysplasia (P = 0.02). No BRAF(V600E) mutation or loss of MLH1 expression was observed. CONCLUSIONS: To our knowledge, this is the first series reporting serrated adenoma in the small intestine, a rare subtype of adenomas resembling traditional serrated adenoma with aggressive morphological features. The absence of the BRAF(V600E) mutation does not support a role for the serrated neoplasia pathway in the development of these lesions, as in colorectal serrated polyps.

The TAS1R2 G-protein-coupled receptor is an ambient glucose sensor in skeletal muscle that regulates NAD homeostasis and mitochondrial capacity.

The bioavailability of nicotinamide adenine dinucleotide (NAD) is vital for skeletal muscle health, yet the mechanisms or signals regulating NAD homeostasis remain unclear. Here, we uncover a pathway connecting peripheral glucose sensing to the modulation of muscle NAD through TAS1R2, the sugar-sensing G protein-coupled receptor (GPCR) initially identified in taste perception. Muscle TAS1R2 receptor stimulation by glucose and other agonists induces ERK1/2-dependent phosphorylation and activation of poly(ADP-ribose) polymerase1 (PARP1), a major NAD consumer in skeletal muscle. Consequently, muscle-specific deletion of TAS1R2 (mKO) in male mice suppresses PARP1 activity, elevating NAD levels and enhancing mitochondrial capacity and running endurance. Plasma glucose levels negatively correlate with muscle NAD, and TAS1R2 receptor deficiency enhances NAD responses across the glycemic range, implicating TAS1R2 as a peripheral energy surveyor. These findings underscore the role of GPCR signaling in NAD regulation and propose TAS1R2 as a potential therapeutic target for maintaining muscle health.

A general path for large-scale solubilization of cellular proteins: from membrane receptors to multiprotein complexes.

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format.