The Utility of Unsupervised Machine Learning in Anatomic Pathology.

OBJECTIVES: Developing accurate supervised machine learning algorithms is hampered by the lack of representative annotated datasets. Most data in anatomic pathology are unlabeled and creating large, annotated datasets is a time consuming and laborious process. Unsupervised learning, which does not require annotated data, possesses the potential to assist with this challenge. This review aims to introduce the concept of unsupervised learning and illustrate how clustering, generative adversarial networks (GANs) and autoencoders have the potential to address the lack of annotated data in anatomic pathology. METHODS: A review of unsupervised learning with examples from the literature was carried out. RESULTS: Clustering can be used as part of semisupervised learning where labels are propagated from a subset of annotated data points to remaining unlabeled data points in a dataset. GANs may assist by generating large amounts of synthetic data and performing color normalization. Autoencoders allow training of a network on a large, unlabeled dataset and transferring learned representations to a classifier using a smaller, labeled subset (unsupervised pretraining). CONCLUSIONS: Unsupervised machine learning techniques such as clustering, GANs, and autoencoders, used individually or in combination, may help address the lack of annotated data in pathology and improve the process of developing supervised learning models.

Strategies to expand peptide functionality through hybridisation with a small molecule component.

Combining different compound classes gives molecular hybrids that can offer access to novel chemical space and unique properties. Peptides provide ideal starting points for such molecular hybrids, which can be easily modified with a variety of molecular entities. The addition of small molecules can improve the potency, stability and cell permeability of therapeutically relevant peptides. Furthermore, they are often applied to create peptide-based tools in chemical biology. In this review, we discuss general methods that allow the discovery of this compound class and highlight key examples of peptide-small molecule hybrids categorised by the application and function of the small molecule entity.

Fluorescent Amino Acid Initiated de novo Cyclic Peptides for the Label-Free Assessment of Cell Permeability*.

The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e. g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and ß-(1-azulenyl)-L-alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.

Zap-Pano: a Photocaged Prodrug of the KDAC Inhibitor Panobinostat.

We report the synthesis and biological evaluation of a light-activated (caged) prodrug of the KDAC inhibitor panobinostat (Zap-Pano). We demonstrate that addition of the 4,5-dimethoxy-2-nitrobenzyl group to the hydroxamic acid oxygen results in an inactive prodrug. In two cancer cell lines we show that photolysis of this compound releases panobinostat and an unexpected carboxamide analogue of panobinostat. Photolysis of Zap-Pano causes an increase in H3K9Ac and H3K18Ac, consistent with KDAC inhibition, in an oesophageal cancer cell line (OE21). Irradiation of OE21 cells in the presence of Zap-Pano results in apoptotic cell death. This compound is a useful research tool, allowing spatial and temporal control over release of panobinostat.

Development and pre-clinical testing of a novel hypoxia-activated KDAC inhibitor.

Tumor hypoxia is associated with therapy resistance and poor patient prognosis. Hypoxia-activated prodrugs, designed to selectively target hypoxic cells while sparing normal tissue, represent a promising treatment strategy. We report the pre-clinical efficacy of 1-methyl-2-nitroimidazole panobinostat (NI-Pano, CH-03), a novel bioreductive version of the clinically used lysine deacetylase inhibitor, panobinostat. NI-Pano was stable in normoxic (21% O2) conditions and underwent NADPH-CYP-mediated enzymatic bioreduction to release panobinostat in hypoxia (<0.1% O2). Treatment of cells grown in both 2D and 3D with NI-Pano increased acetylation of histone H3 at lysine 9, induced apoptosis, and decreased clonogenic survival. Importantly, NI-Pano exhibited growth delay effects as a single agent in tumor xenografts. Pharmacokinetic analysis confirmed the presence of sub-micromolar concentrations of panobinostat in hypoxic mouse xenografts, but not in circulating plasma or kidneys. Together, our pre-clinical results provide a strong mechanistic rationale for the clinical development of NI-Pano for selective targeting of hypoxic tumors.

The cytopathologist's role in developing and evaluating artificial intelligence in cytopathology practice.

Artificial intelligence (AI) technologies have the potential to transform cytopathology practice, and it is important for cytopathologists to embrace this and place themselves at the forefront of implementing these technologies in cytopathology. This review illustrates an archetypal AI workflow from project conception to implementation in a diagnostic setting and illustrates the cytopathologist's role and level of involvement at each stage of the process. Cytopathologists need to develop and maintain a basic understanding of AI, drive decisions regarding the development and implementation of AI in cytopathology, participate in the generation of datasets used to train and evaluate AI algorithms, understand how the performance of these algorithms is assessed, participate in the validation of these algorithms (either at a regulatory level or in the laboratory setting), and ensure continuous quality assurance of algorithms deployed in a diagnostic setting. In addition, cytopathologists should ensure that these algorithms are developed, trained, tested and deployed in an ethical manner. Cytopathologists need to become informed consumers of these AI algorithms by understanding their workings and limitations, how their performance is assessed and how to validate and verify their output in clinical practice.

Extraoral plasmablastic lymphomas in a high human immunodeficiency virus endemic area.

AIMS: Plasmablastic lymphoma (PBL) occurs mainly in immunocompromised individuals, usually secondary to human immunodeficiency virus (HIV) infection. It classically occurs intraorally, but has been described in extraoral locations. The aim of this study was to define the immunophenotype and Epstein-Barr virus (EBV) status in a large single-centre cohort of extraoral PBL (EPBL) in South Africa, a high-prevalence HIV setting. METHODS AND RESULTS: This retrospective study of 45 EPBLs included patients' age, gender, race, HIV status, and site. Cases were reviewed histologically, and classified morphologically as pure plasmablastic or plasmablastic with plasmacytic differentiation, and assessed immunohistochemically with antibodies against CD45, CD20, CD79a, PAX5, CD138, MUM1/IRF4, BLIMP1, VS38c, Ki67, bcl-6, CD10, cyclin D1, and human herpesvirus-8, by the use of standard automated procedures. EBV was assessed by the use of chromogenic in-situ hybridisation. Tumours were assessed with a fluorescence in-situ hybridisation (FISH) MYC break-apart probe. Twenty-seven PBLs showed pure plasmablastic morphology, and 18 showed plasmacytic differentiation. The male/female ratio was 1.5:1. The anus was the favoured extraoral site (31.1%), followed by lymph nodes (15.6%). All 29 patients with known HIV status were HIV-positive. The immunohistochemical profile recapitulated that reported for oral PBLs and EPBLs in HIV-positive and HIV-negative patients. EBV was positive in 92.5% of PBLs. FISH analysis showed MYC rearrangement in 48% of cases. CONCLUSION: This study showed a strong association of EPBLs with HIV and EBV infection, similarly to the previously described oral PBL. The strong EBV association together with other clinicopathological parameters and an immunohistochemical profile that includes CD45, CD20, MUM1/IRF4, CD138 and Ki67 may be used in distinguishing PBL from diffuse large B-cell lymphoma and plasma cell myeloma.

One-Pot Asymmetric Synthesis of Alkylidene 1-Alkylindan-1-ols Using Brønsted Acid and Palladium Catalysis.

A one-pot catalytic enantioselective allylboration/Mizoroki-Heck reaction of 2-bromoaryl ketones has been developed for the asymmetric synthesis of 3-methyleneindanes bearing a tertiary alcohol center. Brønsted acid-catalyzed allylboration with a chiral BINOL derivative was followed by a palladium-catalyzed Mizoroki-Heck cyclization, resulting in selective formation of the exo-alkene. This novel protocol provides a concise and scalable approach to 1-alkyl-3-methyleneindan-1-ols in high enantiomeric ratios (up to 96:4 er). The potential of these compounds as chiral building blocks was demonstrated with efficient transformation to optically active diol and amino alcohol scaffolds.

Clinical Advances of Hypoxia-Activated Prodrugs in Combination With Radiation Therapy.

With the increasing incidence of cancer worldwide, the need for specific, effective therapies is ever more urgent. One example of targeted cancer therapeutics is hypoxia-activated prodrugs (HAPs), also known as bioreductive prodrugs. These prodrugs are inactive in cells with normal oxygen levels but in hypoxic cells (with low oxygen levels) undergo chemical reduction to the active compound. Hypoxia is a common feature of solid tumors and is associated with a more aggressive phenotype and resistance to all modes of therapy. Therefore, the combination of radiation therapy and bioreductive drugs presents an attractive opportunity for synergistic effects, because the HAP targets the radiation-resistant hypoxic cells. Hypoxia-activated prodrugs have typically been precursors of DNA-damaging agents, but a new generation of molecularly targeted HAPs is emerging. By targeting proteins associated with tumorigenesis and survival, these compounds may result in greater selectivity over healthy tissue. We review the clinical progress of HAPs as adjuncts to radiation therapy and conclude that the use of HAPs alongside radiation is vastly underexplored at the clinical level.

Characterization of HSP90 isoforms in transformed bovine leukocytes infected with Theileria annulata.

HSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata-infected counterpart was utilized to test the effects of geldanamycin and the derivative 17-AAG. T. annulata-infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17-AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation.

Intermolecular Aryl C-H Amination through Sequential Iron and Copper Catalysis.

A mild, efficient and regioselective method for para-amination of activated arenes has been developed through a combination of iron and copper catalysis. A diverse range of products were obtained from an operationally simple one-pot, two-step procedure involving bromination of the aryl substrate with the powerful Lewis acid iron(III) triflimide, followed by a copper(I)-catalysed N-arylation reaction. This two-step dehydrogenative process for the regioselective coupling of aromatic C-H bonds with non-activated amines was applicable to anisole-, phenol-, aniline- and acetanilide-type aryl compounds. Importantly, the arene substrates were used as the limiting reagent and required no protecting-group manipulations during the transformation.

Synthesis of Allylic Amide Functionalized 2H-Chromenes and Coumarins Using a One-Pot Overman Rearrangement and Gold(I)-Catalyzed Hydroarylation.

A four-step synthesis of allylic trichloroacetimidates bearing a 2-proparyloxyaryl group has been developed from readily available 2-hydroxybenzaldehydes, and these have been used for the preparation of allylic amide derived 2H-chromenes using an Overman rearrangement and a 6-endo-dig hydroarylation. High yields of the 2H-chromenes were achieved using a stepwise approach involving an Overman rearrangement under thermal conditions followed by a hydroarylation reaction with a gold(I) triflimide catalyst. An alternative method where both reactions were performed as a one-pot process was also developed and instead used a gold(I) chloride catalyst activated by silver(I) hexafluoroantimonate for the cycloisomerization step. The allylic amide derived 2H-chromenes were converted to the corresponding coumarin analogues by a pyridinium dichromate (PDC)-mediated chemoselective allylic oxidation.

Synthesis of 5-Amino-2,5-dihydro-1H-benzo[b]azepines Using a One-Pot Multibond Forming Process.

Rapid access to allylic trichloroacetimidates bearing a 2-allylaminoaryl group from readily available 2-iodoanilines combined with a one-pot multibond forming process has allowed the efficient synthesis of a series of 5-amino-2,5-dihydro-1H-benzo[b]azepines. The potential of these compounds as synthetic building blocks was demonstrated by the preparation of a late-stage intermediate of the hyponatremia agent, mozavaptan.

Enantioselective Synthesis of 3-Methyleneindan-1-ols via a One-Pot Allylboration-Heck Reaction of 2-Bromobenzaldehydes.

A novel, one-pot allylboration-Heck reaction of 2-bromobenzaldehydes has been developed for the general and efficient synthesis of 3-methyleneindan-1-ols. Modification of the one-pot procedure to include chiral Brønsted acid catalyzed allylation has allowed the preparation of these building blocks in high enantioselectivity and excellent yields.

One-Pot Synthesis of 5-Amino-2,5-dihydro-1-benzoxepines: Access to Pharmacologically Active Heterocyclic Scaffolds.

A one-pot multibond-forming process involving a thermally mediated Overman rearrangement and a ring closing metathesis reaction of allylic trichloroacetimidates bearing a 2-allyloxyaryl group has been developed for the synthesis of 5-amino-substituted 2,5-dihydro-1-benzoxepines. Chemoselective reduction and functionalization of these compounds allowed access to a range of pharmacologically active 5-amino-2,3,4,5-tetrahydro-1-benzoxepine scaffolds.

Stereodivergent organocatalytic intramolecular Michael addition/lactonization for the asymmetric synthesis of substituted dihydrobenzofurans and tetrahydrofurans.

A stereodivergent asymmetric Lewis base catalyzed Michael addition/lactonization of enone acids into substituted dihydrobenzofuran and tetrahydrofuran derivatives is reported. Commercially available (S)-(-)-tetramisole hydrochloride gives products with high syn diastereoselectivity in excellent enantioselectivity (up to 99:1 d.r.syn/anti , 99 % eesyn ), whereas using a cinchona alkaloid derived catalyst gives the corresponding anti-diastereoisomers as the major product (up to 10:90 d.r.syn/anti , 99 % eeanti ).

Preparation of amino-substituted indenes and 1,4-dihydronaphthalenes using a one-pot multireaction approach: total synthesis of oxybenzo[c]phenanthridine alkaloids.

Allylic trichloroacetimidates bearing a 2-vinyl or 2-allylaryl group have been designed as substrates for a one-pot, two-step multi-bond-forming process leading to the general preparation of aminoindenes and amino-substituted 1,4-dihydronaphthalenes. The synthetic utility of the privileged structures formed from this one-pot process was demonstrated with the total synthesis of four oxybenzo[c]phenanthridine alkaloids, oxychelerythrine, oxysanguinarine, oxynitidine, and oxyavicine. An intramolecular biaryl Heck coupling reaction, catalyzed using the Hermann-Beller palladacycle was used to effect the key step during the synthesis of the natural products.

Preparation of anti-vicinal amino alcohols: asymmetric synthesis of D-erythro-sphinganine, (+)-spisulosine, and D-ribo-phytosphingosine.

Two variations of the Overman rearrangement have been developed for the highly selective synthesis of anti-vicinal amino alcohol natural products. A MOM ether-directed palladium(II)-catalyzed rearrangement of an allylic trichloroacetimidate was used as the key step for the preparation of the protein kinase C inhibitor D-erythro-sphinganine and the antitumor agent (+)-spisulosine, whereas the Overman rearrangement of chiral allylic trichloroacetimidates generated by the asymmetric reduction of an α,ß-unsaturated methyl ketone allowed rapid access both to D-ribo-phytosphingosine and L-arabino-phytosphingosine.

Provision of servo-controlled cooling during neonatal transport.

INTRODUCTION: Therapeutic hypothermia is a time critical intervention for infants who have experienced a hypoxic-ischaemic event. Previously reported methods of cooling during transport do not demonstrate the same stability achieved in the neonatal unit. The authors developed a system which allowed provision of servo-controlled cooling throughout transport, and present their first year's experience. METHODS: Retrospective review of routinely collected patient data. RESULTS: 14 out-born infants were referred for cooling during a 12-month period. Nine infants were managed with the servo-controlled system during transport. Cooling was commenced in all infants before 6 h of life. Median time from team arrival to the infant having a temperature in the target range (33-34°C) was 45 min. Median temperature during transfer was 33.5°C (range 33-34°C). Temperature on arrival at the cooling centre ranged from 33.4°C to 33.8°C. CONCLUSION: Servo-controlled cooling during transport is feasible and provides an optimal level of thermal control.

Oxygen targeting in preterm infants using the Masimo SET Radical pulse oximeter.

BACKGROUND: A pretrial clinical improvement project for the BOOST-II UK trial of oxygen saturation targeting revealed an artefact affecting saturation profiles obtained from the Masimo Set Radical pulse oximeter. METHODS: Saturation was recorded every 10 s for up to 2 weeks in 176 oxygen dependent preterm infants in 35 UK and Irish neonatal units between August 2006 and April 2009 using Masimo SET Radical pulse oximeters. Frequency distributions of % time at each saturation were plotted. An artefact affecting the saturation distribution was found to be attributable to the oximeter's internal calibration algorithm. Revised software was installed and saturation distributions obtained were compared with four other current oximeters in paired studies. RESULTS: There was a reduction in saturation values of 87-90%. Values above 87% were elevated by up to 2%, giving a relative excess of higher values. The software revision eliminated this, improving the distribution of saturation values. In paired comparisons with four current commercially available oximeters, Masimo oximeters with the revised software returned similar saturation distributions. CONCLUSIONS: A characteristic of the software algorithm reduces the frequency of saturations of 87-90% and increases the frequency of higher values returned by the Masimo SET Radical pulse oximeter. This effect, which remains within the recommended standards for accuracy, is removed by installing revised software (board firmware V4.8 or higher). Because this observation is likely to influence oxygen targeting, it should be considered in the analysis of the oxygen trial results to maximise their generalisability.