Development of Centrifugal Partition Chromatography for the Purification of Antibody-Drug Conjugates.

Monoclonal antibody-drug conjugates (ADCs) are an expanding therapeutic class of biomolecules for which relatively few analytical and preparative separation options exist. Purification of ADCs with a specific drug antibody ratio is even more challenging. We report the first application of countercurrent separation (CCS) to this problem. An ADC mimic was successfully chromatographed using an aqueous two-phase system (ATPS) consisting of PEG 1000/sodium citrate pH 7.5/water, 17.75/17.75/64.50 (w/w/w). Notably, different partition coefficients (K) in this ATPS for the ADC mimic (0.09 < K < 0.16) and its monoclonal antibody backbone, IgG (0.16 < K < 0.27), were observed using CCS. Differential elution behavior of such high-molecular-weight biomolecules, 146,441 vs. ∼150,000 Da, using CCS has no precedent. The results provide a proof of concept for further exploration of the application of ATPSs and CCS to the separation of ADCs.

Effect of Relative Humidity on Transfer of Aerosol-Deposited Artificial and Human Saliva from Surfaces to Artificial Finger-Pads.

Surface to hand transfer of viruses represents a potential mechanism for human exposure. An experimental process for evaluating the touch transfer of aerosol-deposited material is described based on controlling surface, tribological, and soft matter components of the transfer process. A range of high-touch surfaces were evaluated. Under standardized touch parameters (15 N, 1 s), relative humidity (RH) of the atmosphere around the contact transfer event significantly influenced transfer of material to the finger-pad. At RH < 40%, transfer from all surfaces was <10%. Transfer efficiency increased markedly as RH increased, reaching a maximum of approximately 50%. The quantity of material transferred at specific RHs above 40% was also dependent on roughness of the surface material and the properties of the aerosol-deposited material. Smooth surfaces, such as melamine and stainless steel, generated higher transfer efficiencies compared to those with textured roughness, such as ABS pinseal and KYDEX® plastics. Pooled human saliva was transferred at a lower rate compared to artificial saliva, indicating the role of rheological properties. The artificial saliva data were modeled by non-linear regression and the impact of environmental humidity and temperature were evaluated within a Quantitative Microbial Risk Assessment model using SARS-CoV-2 as an example. This illustrated that the trade-off between transfer efficiency and virus survival may lead to the highest risks of fomite transmissions in indoor environments with higher humidity.

GCSscore: an R package for differential gene expression analysis in Affymetrix/Thermo-Fisher whole transcriptome microarrays.

BACKGROUND: Despite the increasing use of RNAseq for transcriptome analysis, microarrays remain a widely-used methodology for genomic studies. The latest generation of Affymetrix/Thermo-Fisher microarrays, the ClariomD/XTA and ClariomS array, provide a sensitive and facile method for complex transcriptome expression analysis. However, existing methods of analysis for these high-density arrays do not leverage the statistical power contained in having multiple oligonucleotides representing each gene/exon, but rather summarize probes into a single expression value. We previously developed a methodology, the Sscore algorithm, for probe-level identification of differentially expressed genes (DEGs) between treatment and control samples with oligonucleotide microarrays. The Sscore algorithm was validated for sensitive detection of DEGs by comparison with existing methods. However, the prior version of the Sscore algorithm and a R-based implementation software, sscore, do not function with the latest generations of Affymetrix/Fisher microarrays due to changes in microarray design that eliminated probes previously used for estimation of non-specific binding. RESULTS: Here we describe the GCSscore algorithm, which utilizes the GC-content of a given oligonucleotide probe to estimate non-specific binding using antigenomic background probes found on new generations of arrays. We implemented this algorithm in an improved GCSscore R package for analysis of modern oligonucleotide microarrays. GCSscore has multiple methods for grouping individual probes on the ClariomD/XTA chips, providing the user with differential expression analysis at the gene-level and the exon-level. By utilizing the direct probe-level intensities, the GCSscore algorithm was able to detect DEGs under stringent statistical criteria for all Clariom-based arrays. We demonstrate that for older 3'-IVT arrays, GCSscore produced very similar differential gene expression analysis results compared to the original Sscore method. However, GCSscore functioned well for both the ClariomS and ClariomD/XTA newer microarrays and outperformed existing analysis approaches insofar as the number of DEGs and cognate biological functions identified. This was particularly striking for analysis of the highly complex ClariomD/XTA based arrays. CONCLUSIONS: The GCSscore package represents a powerful new application for analysis of the newest generation of oligonucleotide microarrays such as the ClariomS and ClariomD/XTA arrays produced by Affymetrix/Fisher.

Campafungins: Inhibitors of Candida albicans and Cryptococcus neoformans Hyphal Growth.

Campafungin A is a polyketide that was recognized in the Candida albicans fitness test due to its antiproliferative and antihyphal activity. Its mode of action was hypothesized to involve inhibition of a cAMP-dependent PKA pathway. The originally proposed structure appeared to require a polyketide assembled in a somewhat unusual fashion. However, structural characterization data were never formally published. This background stimulated a reinvestigation in which campafungin A and three closely related minor constituents were purified from fermentations of a strain of the ascomycete fungus Plenodomus enteroleucus. Labeling studies, along with extensive NMR analysis, enabled assignment of a revised structure consistent with conventional polyketide synthetic machinery. The structure elucidation of campafungin A and new analogues encountered in this study, designated here as campafungins B, C, and D, is presented, along with a proposed biosynthetic route. The antimicrobial spectrum was expanded to methicillin-resistant Staphylococcus aureus, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, and Schizosaccharomyces pombe, with MICs ranging as low as 4-8 µg mL-1 in C. neoformans. Mode-of-action studies employing libraries of C. neoformans mutants indicated that multiple pathways were affected, but mutants in PKA/cAMP pathways were unaffected, indicating that the mode of action was distinct from that observed in C. albicans.

How often does computed tomography (CT) of the brain demonstrate a cause for psychosis? A 7-year retrospective study at a tertiary metropolitan hospital.

OBJECTIVES: The aim of the study was to determine the diagnostic yield of computed tomography (CT) of the brain for the investigation of psychosis. METHODS: CT brain requests describing psychosis over a 7-year period at a 500-bed major metropolitan hospital were identified retrospectively. Patients were excluded if they were aged greater than 50 years or if the CT request described focal neurological findings on examination, trauma/falls or known brain tumour, demyelinating disorder, encephalopathy, seizure disorder, congenital brain anomaly, stroke or traumatic brain injury. RESULTS: A total of 805 patients meeting the inclusion and exclusion criteria were identified, representing the largest published study on this topic. Only 0.4% of patients (3 out of 805) had a potential cause for psychosis demonstrated on CT. None of these patients had their management altered as a result. An additional 0.6% of patients (5 out of 805) had significant pathology that was deemed unrelated to their psychosis. CONCLUSIONS: The diagnostic value of CT in the setting of psychosis was found to be extremely low in patients meeting the inclusion and exclusion criteria. Given the risk of ionising radiation and the expenditure of time and cost, more judicious use of CT is suggested.

Intermittent Ethanol during Adolescence Leads to Lasting Behavioral Changes in Adulthood and Alters Gene Expression and Histone Methylation in the PFC.

Adolescents primarily consume alcohol in binges, which can be particularly harmful to the developing frontal cortex and increase risk for an adult alcohol use disorder. We conducted a study investigating immediate and long lasting changes to the prefrontal cortex (PFC) transcriptome to determine the molecular mechanisms underlying adult ethanol behavioral sensitivity following binge ethanol in adolescence. DBA/2J mice were orally dosed with 4 g/kg ethanol intermittently from day 29 to 42. Adolescent mice were tested for anxiety-like behavior and ethanol sensitivity using the loss of righting reflex task. As adults, mice were tested for cognitive changes using the novel object recognition task, ethanol-induced anxiolysis and ethanol sensitivity. Adolescent binge ethanol altered ethanol sensitivity in young mice and led to lasting memory deficits in the object recognition test and greater ethanol sensitivity in adulthood. Using genomic profiling of transcripts in the PFC, we found that binge ethanol reduced myelin-related gene expression and altered chromatin modifying genes involved in histone demethylation at H3K9 and H3K36. We hypothesize that ethanol's actions on histone methylation may be a switch for future transcriptional changes that underlie the behavioral changes lasting into adulthood.

The allostatic impact of chronic ethanol on gene expression: A genetic analysis of chronic intermittent ethanol treatment in the BXD cohort.

The transition from acute to chronic ethanol exposure leads to lasting behavioral and physiological changes such as increased consumption, dependence, and withdrawal. Changes in brain gene expression are hypothesized to underlie these adaptive responses to ethanol. Previous studies on acute ethanol identified genetic variation in brain gene expression networks and behavioral responses to ethanol across the BXD panel of recombinant inbred mice. In this work, we have performed the first joint genetic and genomic analysis of transcriptome shifts in response to chronic intermittent ethanol (CIE) by vapor chamber exposure in a BXD cohort. CIE treatment is known to produce significant and sustained changes in ethanol consumption with repeated cycles of ethanol vapor. Using Affymetrix microarray analysis of prefrontal cortex (PFC) and nucleus accumbens (NAC) RNA, we compared CIE expression responses to those seen following acute ethanol treatment, and to voluntary ethanol consumption. Gene expression changes in PFC and NAC after CIE overlapped significantly across brain regions and with previously published expression following acute ethanol. Genes highly modulated by CIE were enriched for specific biological processes including synaptic transmission, neuron ensheathment, intracellular signaling, and neuronal projection development. Expression quantitative trait locus (eQTL) analyses identified genomic loci associated with ethanol-induced transcriptional changes with largely distinct loci identified between brain regions. Correlating CIE-regulated genes to ethanol consumption data identified specific genes highly associated with variation in the increase in drinking seen with repeated cycles of CIE. In particular, multiple myelin-related genes were identified. Furthermore, genetic variance in or near dynamin3 (Dnm3) on Chr1 at ∼164 Mb may have a major regulatory role in CIE-responsive gene expression. Dnm3 expression correlates significantly with ethanol consumption, is contained in a highly ranked functional group of CIE-regulated genes in the NAC, and has a cis-eQTL within a genomic region linked with multiple CIE-responsive genes.

Imaging of tarsal navicular stress injury with a focus on MRI: A pictorial essay.

Predominantly diagnosed in athletes, stress fracture of the tarsal navicular is becoming increasingly recognised by clinicians as a cause of midfoot pain. Delayed diagnosis can increase the significant morbidity associated with this condition. Consequently the role of MRI is increasing, given the potential to identify a stress reaction in the navicular prior to the development of a discrete stress fracture. It is necessary for radiologists to be familiar with the typical and atypical appearances of this important condition.

Automated solvent system screening for the preparative countercurrent chromatography of pharmaceutical discovery compounds.

A fully automated countercurrent chromatography system has been constructed to rapidly screen the commonly used heptane/ethyl acetate/methanol/water solvent system series and translate the results to preparative scale separations. The system utilizes "on-demand" preparation of the heptane/ethyl acetate/methanol/water solvent system upper and lower phases. Elution-extrusion countercurrent chromatography was combined with non-dynamic equilibrium injection reducing the screening time for each heptane/ethyl acetate/methanol/water system to 17 min. The result enabled solvent system development to be reduced to under 2 h. The countercurrent chromatography system was interfaced with a mass spectrometer to allow selective detection of target components in crude medicinal chemistry reaction mixtures. Mass-directed preparative countercurrent chromatography purification was demonstrated for the first time using a synthetic tetrazole epoxide derived from a routine medicinal chemistry support workflow.

Associations Between Antral Ovarian Follicle Dynamics and Hormone Production Throughout the Menstrual Cycle as Women Age.

BACKGROUND: The physiological origins of age-related changes in hormone production during the menstrual cycle are uncertain. OBJECTIVE: The objective of the study was to test the hypothesis that changes in antral follicle dynamics are associated with changes in hormone production as women age. METHODS: A prospective, observational study was conducted in ovulatory women of midreproductive age (MRA; 18-35 y; n = 10) and advanced reproductive age (ARA; 45-55 y; n = 20). The numbers and diameters of all follicles of 2 mm or greater were quantified ultrasonographically every 1-3 days for one interovulatory interval; the growth profiles of individually identified follicles of 4 mm or greater were tabulated. Blood samples were assayed for FSH, LH, estradiol, progesterone, inhibin A and B, and anti-Mullerian hormone. RESULTS: Fifty percent of women in both the MRA and ARA groups developed one to two luteal-phase dominant follicles (LPDFs). MRA women with typical LPDFs had greater luteal-phase inhibin B (44.2 vs 17.0 ng/L) and estradiol (91.3 vs 51.7 ng/L) compared with those without LPDFs (P < .05). Luteal-phase estradiol was greater (184 vs 79 ng/L), inhibin B was greater (25.3 vs 12.7 ng/L), and progesterone was lower (6.98 vs 13.8 µg/L) in ARA women with atypical vs no LPDFs (P < .01). CONCLUSION: Changes in antral follicle dynamics are associated with changes in hormone production as women age. The development of LPDFs in women of MRA was associated with elevated luteal-phase estradiol. A similar but exaggerated elevation in late luteal-early follicular-phase estradiol, accompanied by lower progesterone, was observed in ARA women with atypically large and persistent LPDFs.

Chemical Genomics-Based Antifungal Drug Discovery: Targeting Glycosylphosphatidylinositol (GPI) Precursor Biosynthesis.

Steadily increasing antifungal drug resistance and persistent high rates of fungal-associated mortality highlight the dire need for the development of novel antifungals. Characterization of inhibitors of one enzyme in the GPI anchor pathway, Gwt1, has generated interest in the exploration of targets in this pathway for further study. Utilizing a chemical genomics-based screening platform referred to as the Candida albicans fitness test (CaFT), we have identified novel inhibitors of Gwt1 and a second enzyme in the glycosylphosphatidylinositol (GPI) cell wall anchor pathway, Mcd4. We further validate these targets using the model fungal organism Saccharomyces cerevisiae and demonstrate the utility of using the facile toolbox that has been compiled in this species to further explore target specific biology. Using these compounds as probes, we demonstrate that inhibition of Mcd4 as well as Gwt1 blocks the growth of a broad spectrum of fungal pathogens and exposes key elicitors of pathogen recognition. Interestingly, a strong chemical synergy is also observed by combining Gwt1 and Mcd4 inhibitors, mirroring the demonstrated synthetic lethality of combining conditional mutants of GWT1 and MCD4. We further demonstrate that the Mcd4 inhibitor M720 is efficacious in a murine infection model of systemic candidiasis. Our results establish Mcd4 as a promising antifungal target and confirm the GPI cell wall anchor synthesis pathway as a promising antifungal target area by demonstrating that effects of inhibiting it are more general than previously recognized.

A general method for the separation of triphenylphosphine oxide and reaction products using high performance countercurrent chromatography.

A standardised separation methodology was developed for the purification of crude reaction mixtures containing triphenylphosphine oxide (TPPO) using high performance countercurrent chromatography (HPCCC). A solvent system consisting of hexane/ethyl acetate/methanol/water (5:6:5:6) was used in 1 column volume elution-extrusion mode. The HPCCC methodology was compared with classical RP HPLC purification using a set of 12 representative Mitsunobu reaction mixtures. HPCCC was seen to yield a 65% increase in the average recovery of the target component whilst providing similar final target purities to those obtained by HPLC. By eliminating the need for method development for individual samples, the HPCCC methodology described within provides a simple and efficient means for the purification of the entire family of TPPO-containing reaction products.

Adhesive capsulitis: review of imaging and treatment.

Adhesive capsulitis is one of the most common conditions affecting the shoulder; however, early clinical diagnosis can be challenging. Treatment is most effective when commenced prior to the onset of capsular thickening and contracture; consequently, the role of imaging is increasing. The aim of this review is to demonstrate the typical imaging appearances of adhesive capsulitis and to examine some of the evidence regarding each of these imaging modalities. An evaluation of the various management options available to the clinician is also presented.

Use of detergent-based buffers allows detection of precursor inhibin forms in an immunoassay format.

Inhibin ELISAs are used in monitoring aspects of reproductive function, however these assays are based on the measurements of the mature 30kDa inhibin forms and not precursor forms. In conventional ELISA formats, the 105kDa unprocessed 'Pro-inhibin' forms are immunologically inactive, but the immunoactivity can be recovered in the presence of detergents. The immunoactivity of Pro-inhibin forms was assessed in the presence of a range of detergents utilising antibodies to the α-, ßA- and ßB-subunits of inhibin. In contrast to mature forms, unprocessed inhibin forms showed a 10-40 fold increase in inhibin A and total inhibin immunoactivities under optimised detergent (0.5% SDS/2% Triton X-100) conditions. The suppressed immunoactivity of the Pro-inhibin forms in these immunoassays was attributed to steric hindrance by the respective ßA- and α-subunit prodomains. This study details a detergent-based immunoassay that allows detection of previously undetectable precursor inhibin forms.

Scalable technology for the extraction of pharmaceutics: outcomes from a 3 year collaborative industry/academia research programme.

This paper reports on some of the key outcomes of a 3 year £1.5m Technology Strategy Board (TSB) funded research programme to develop a small footprint, versatile, counter-current chromatography purification technology and methodology which can be operated at a range of scales in both batch and continuous modes and that can be inserted into existing process plant and systems. Our consortium, integrates technology providers (Dynamic Extractions) and the scientific development team (Brunel) with end user needs (GSK & Pfizer), addressing major production challenges aimed at providing flexible, low capital platform technology driving substantial cost efficiency in both drug development and drug manufacturing processes. The aims of the Technology Strategy Board's high value manufacturing programme are described and how the academic/industry community were challenged to instigate step changes in the manufacturing of high value pharmaceuticals. This paper focusses on one of the themes of the TSB research programme, "Generate a Comprehensive Applications Portfolio". It outlines 15 applications from this portfolio that can be published in the public domain and gives four detailed case studies illustrating the range of application of the technology on the separation of (1) isomers, (2) polar compounds, (3) crude mixtures and (4) on the removal of impurities. Two of these case studies that were scaled up demonstrate between 10 and 20% lower solvent usage and were projected to have significant cost savings compared to conventional solid phase silica gel chromatography at procss scale demonstrating that the latest high performance countercurrent chromatography technology is a competitive platform technolgy for the pharmaceutical industry.

Isolation, structure, and biological activity of Phaeofungin, a cyclic lipodepsipeptide from a Phaeosphaeria sp. Using the Genome-Wide Candida albicans Fitness Test.

Phaeofungin (1), a new cyclic depsipeptide isolated from Phaeosphaeria sp., was discovered by application of reverse genetics technology, using the Candida albicans fitness test (CaFT). Phaeofungin is comprised of seven amino acids and a ß,γ-dihydroxy-γ-methylhexadecanoic acid arranged in a 25-membered cyclic depsipeptide. Five of the amino acids were assigned with d-configurations. The structure was elucidated by 2D-NMR and HRMS-MS analysis of the natural product and its hydrolyzed linear peptide. The absolute configuration of the amino acids was determined by Marfey's method by complete and partial hydrolysis of 1. The CaFT profile of the phaeofungin-containing extract overlapped with that of phomafungin (3), another structurally different cyclic lipodepsipeptide isolated from a Phoma sp. using the same approach. Comparative biological characterization further demonstrated that these two fungal lipodepsipeptides are functionally distinct. While phomafungin was potentiated by cyclosporin A (an inhibitor of the calcineurin pathway), phaeofungin was synergized with aureobasidin A (2) (an inhibitor of the sphingolipid biosynthesis) and to some extent caspofungin (an inhibitor of glucan synthase). Furthermore, phaeofungin caused ATP release in wild-type C. albicans strains but phomafungin did not. It showed modest antifungal activity against C. albicans (MIC 16-32 µg/mL) and better activity against Aspergillus fumigatus (MIC 8-16 µg/mL) and Trichophyton mentagrophytes (MIC 4 µg/mL). The linear peptide was inactive, suggesting that the macrocyclic depsipeptide ring is essential for target engagement and antifungal activity.

Isolation of bitter acids from hops (Humulus lupulus L.) using countercurrent chromatography.

Commercially available hops (Humulus lupulus L.) bitter acid extracts contain a mixture of three major congeners (co-, n-, and ad-) in addition to cis/trans diastereomers for each congener. Individual isomerized α-acids were obtained by the consecutive application of two separate countercurrent chromatography methods. First, individual isomerized α-acid congeners as a mixture of cis/trans diastereomers were obtained using a solvent system consisting of hexane and aqueous buffer. The second purification, capable of separating cis/trans diastereomers, was accomplished using a quaternary solvent system; an alternative procedure using ß-cyclodextrin followed by countercurrent chromatography was also investigated. The NaBH(4) reduction of the purified isomerized α-acid compounds followed by countercurrent chromatography purification resulted in individual ρ iso α-acids (>95%). Similarly, catalytic hydrogenation of the purified isomerized α-acid compounds followed by countercurrent chromatography purification produced individual tetrahydro isomerized α-acids (>95%). Reported herein is a widely applicable approach that focuses on three critical variables--solvent system composition, pH, and buffer-to-sample ratio--that enable the efficient purification of individual bitter acids (≥95%) from commercially available hops extracts.

Comparison of preparative reversed phase liquid chromatography and countercurrent chromatography for the kilogram scale purification of crude spinetoram insecticide.

Reversed phase HPLC (RP-HPLC) and high performance countercurrent chromatography (HPCCC) were compared for the pilot scale purification of two semi-synthetic spinosyns, spinetoram-J and spinetoram-L, the major components of the commercial insecticide spinetoram. Two, independently performed, 1 kg, purification campaigns were compared. Each method resulted in the isolation of both components at a purity of >97% and yields for spinetoram-J and spinetoram-L of >93% and ≥ 63% of theoretical, respectively. The HPCCC process produced a 2-fold higher throughput and consumed approximately 70% less solvent than preparative scale RP-HPLC, the volume of product containing fractions from HPCCC amounted to 7% of that produced by HPLC and so required much less post-run processing.

Isolation, structure, and biological activities of Fellutamides C and D from an undescribed Metulocladosporiella (Chaetothyriales) using the genome-wide Candida albicans fitness test.

In a whole-cell mechanism of action (MOA)-based screening strategy for discovery of antifungal agents, Candida albicans was used, followed by testing of active extracts in the C. albicans fitness test (CaFT), which provides insight into the mechanism of action. A fermentation extract of an undescribed species of Metulocladosporiella that inhibited proteasome activity in a C. albicans fitness test was identified. The chemical genomic profile of the extract contained hypersensitivity of heterozygous deletion strains (strains that had one of the genes of the diploid genes knocked down) of genes represented by multiple subunits of the 25S proteasome. Two structurally related peptide aldehydes, named fellutamides C and D, were isolated from the extract. Fellutamides were active against C. albicans and Aspergillus fumigatus with MICs ranging from 4 to 16 µg/mL and against fungal proteasome (IC50 0.2 µg/mL). Both compounds showed proteasome activity against human tumor cell lines, potently inhibiting the growth of PC-3 prostate carcinoma cells, but not A549 lung carcinoma cells. In PC-3 cells compound treatment produced a G2M cell cycle block and induced apoptosis. Preliminary SAR studies indicated that the aldehyde group is critical for the antifungal activity and that the two hydroxy groups are quantitatively important for potency.