Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Substantial improvement of histopathological diagnosis by whole-slide image-based remote consultation.

Consultation by subspecialty experts is the most common mode of rendering diagnosis in challenging cases in pathological practice. Our study aimed to highlight the diagnostic benefits of whole-slide image (WSI)-based remote consultation. We obtained diagnostically challenging cases from two institutions from the years 2010 and 2013, with histological diagnoses that contained keywords "probable," "suggestive," "suspicious," "inconclusive," and "uncertain." A total of 270 cases were selected for remote consultation using WSIs scanned at 40 × . The consultation process consisted of three rounds: the first and second rounds each with 12 subspecialty experts and the third round with six multi-expertise senior pathologists. The first consultation yielded 44% concordance, and a change in diagnosis occurred in 56% of cases. The most frequent change was from inconclusive to definite diagnosis (30%), followed by minor discordance (14%), and major discordance (12%). Out of the 70 cases which reached the second round, 31 cases showed discrepancy between the two consultants. For these 31 cases, a consensus diagnosis was provided by six multi-expertise senior pathologists. Combining all WSI-based consultation rounds, the original inconclusive diagnosis was changed in 140 (52%) out of 266 cases. Among these cases, 80 cases (30%) upgraded the inconclusive diagnosis to a definite diagnosis, and 60 cases (22%) changed the diagnosis with major or minor discordance, accounting for 28 cases (10%) and 32 cases (12%), respectively. We observed significant improvement in the pathological diagnosis of difficult cases by remote consultation using WSIs, which can further assist in patient healthcare. A post-study survey highlighted various benefits of WSI-based consults.

Whole-slide imaging at primary pathological diagnosis: Validation of whole-slide imaging-based primary pathological diagnosis at twelve Japanese academic institutes.

Several reports have demonstrated the use of whole-slide imaging (WSI) for primary pathological diagnosis, but no such studies have been published from Asia. We retrospectively collected 1070 WSI specimens from 900 biopsies and small surgeries conducted in nine hospitals. Nine pathologists, who participated in this study, trained for the College of American Pathologists guidelines, reviewed the specimens and made diagnoses based on digitized, 20× or 40× optically magnified images with a WSI scanner. After a washout interval of over 2 weeks, the same observers reviewed conventional glass slides and diagnosed them by light microscopy. Discrepancies between microscopy- and WSI-based diagnoses were evaluated at the individual institutes, and discrepant cases were further reviewed by all pathologists. Nine diagnoses (0.9%) showed major discrepancies with significant clinical differences between the WSI- and microscopy-based diagnoses, and 37 (3.5%) minor discrepancies occurred without a clinical difference. Eight out of nine diagnoses with a major discrepancy were considered concordant with the microscopy-based diagnoses. No association was observed between the level of discrepancy and the organ type, collection method, or digitized optical magnification. Our results indicate the availability of WSI-based primary diagnosis of biopsies and small surgeries in routine daily practice.