De novo variants in POLR3B cause ataxia, spasticity, and demyelinating neuropathy.

POLR3B encodes the second-largest catalytic subunit of RNA polymerase III, an enzyme involved in transcription. Bi-allelic pathogenic variants in POLR3B are a well-established cause of hypomyelinating leukodystrophy. We describe six unrelated individuals with de novo missense variants in POLR3B and a clinical presentation substantially different from POLR3-related leukodystrophy. These individuals had afferent ataxia, spasticity, variable intellectual disability and epilepsy, and predominantly demyelinating sensory motor peripheral neuropathy. Protein modeling and proteomic analysis revealed a distinct mechanism of pathogenicity; the de novo POLR3B variants caused aberrant association of individual enzyme subunits rather than affecting overall enzyme assembly or stability. We expand the spectrum of disorders associated with pathogenic variants in POLR3B to include a de novo heterozygous POLR3B-related disorder.

Association between ATP luminometry of feeding equipment and environmental and health parameters of preweaned calves on dairy farms.

The objective of this study was to examine the influence of different environmental factors on ATP luminometry measurements of feeding equipment and to investigate associations with health of preweaned calves and the levels of ATP identified through luminometry. On 50 commercial dairy farms in Quebec, Canada, ATP luminometry measurements (in relative light units (RLU)) were obtained using the direct swabbing technique with Hygiena UltraSnap swabs and a liquid rinsing technique with the same swab for automatic milk feeders (AMF), bottles, buckets, esophageal tube feeders (ET), milk replacer, nipples and water. During this visit, environmental factors (including temperature, air draft, humidity, ammonia, and bacterial count) were collected and a clinical examination (including respiratory score and fecal score) was performed for all preweaned calves present at the farm. This process was repeated 4 times in a year, leading to collection of luminometer results, environmental parameters, and overall health of calves for each season per farm. Overall, a difference in luminometer results was seen between the different periods sampled for all feeding equipment (except the ET), milk replacer and water, showing higher RLU values in spring and summer and lower values in autumn and winter. When comparing RLU measurements with environmental factors, only a low to negligible correlation could be found. When feeding equipment was classified as not contaminated or contaminated based on previously described cut-off values, a good agreement within a farm for the different seasons was noticed only for nipples (Gwet's agreement AC1 = 0.64), with a poor to moderate agreement for other feeding equipment. Regarding the different models of nipples, 'Peach Teat' nipples showed higher RLU values compared with 'Merricks' nipples models. An association was seen between the proportion of preweaned calves suffering from diarrhea on the farm and the contamination of AMF based on ATP luminometry (logistic regression estimate = 0.52, P = 0.04). For other feeding equipment, milk replacer and water, no significant associations were found. This study showed that ATP luminometry measurements of feeding equipment from preweaned calves are susceptible to seasonality and type of nipple. Thus, these factors should be taken into consideration when interpreting results. Additionally, an association could be made between diarrhea in preweaned calves and the contamination of AMF based on ATP luminometry, showing the potential clinical importance of this on-farm hygiene assessment tool.

A role for triglyceride lipase brummer in the regulation of sex differences in Drosophila fat storage and breakdown.

Triglycerides are the major form of stored fat in all animals. One important determinant of whole-body fat storage is whether an animal is male or female. Here, we use Drosophila, an established model for studies on triglyceride metabolism, to gain insight into the genes and physiological mechanisms that contribute to sex differences in fat storage. Our analysis of triglyceride storage and breakdown in both sexes identified a role for triglyceride lipase brummer (bmm) in the regulation of sex differences in triglyceride homeostasis. Normally, male flies have higher levels of bmm mRNA both under normal culture conditions and in response to starvation, a lipolytic stimulus. We find that loss of bmm largely eliminates the sex difference in triglyceride storage and abolishes the sex difference in triglyceride breakdown via strongly male-biased effects. Although we show that bmm function in the fat body affects whole-body triglyceride levels in both sexes, in males, we identify an additional role for bmm function in the somatic cells of the gonad and in neurons in the regulation of whole-body triglyceride homeostasis. Furthermore, we demonstrate that lipid droplets are normally present in both the somatic cells of the male gonad and in neurons, revealing a previously unrecognized role for bmm function, and possibly lipid droplets, in these cell types in the regulation of whole-body triglyceride homeostasis. Taken together, our data reveal a role for bmm function in the somatic cells of the gonad and in neurons in the regulation of male-female differences in fat storage and breakdown and identify bmm as a link between the regulation of triglyceride homeostasis and biological sex.

First detection and complete genome sequence of a new potexvirus naturally infecting Adenium obesum.

Here, we report the detection and complete genome sequence of a novel potexvirus, tentatively named "Adenium obesum virus X" (AobVX), isolated from Adenium obesum, that was sent for virus screening at Australian Government post-entry quarantine (PEQ) facilities after being imported into Australia from China. The AobVX genome is 6781 nucleotides in length excluding the poly(A) tail and is predicted to encode conserved potexvirus proteins and sequence motifs across five open reading frames. The RNA-dependent RNA polymerase of this virus shares the highest amino acid sequence similarity with that of nerine potexvirus 1 (58.7% identity) and nerine virus X (58.58% identity). This is the first report of a positive-sense single-stranded RNA virus in A. obesum related to members of the genus Potexvirus in the family Alphaflexiviridae.

The crown-of-thorns starfish genome as a guide for biocontrol of this coral reef pest.

The crown-of-thorns starfish (COTS, the Acanthaster planci species group) is a highly fecund predator of reef-building corals throughout the Indo-Pacific region. COTS population outbreaks cause substantial loss of coral cover, diminishing the integrity and resilience of reef ecosystems. Here we sequenced genomes of COTS from the Great Barrier Reef, Australia and Okinawa, Japan to identify gene products that underlie species-specific communication and could potentially be used in biocontrol strategies. We focused on water-borne chemical plumes released from aggregating COTS, which make the normally sedentary starfish become highly active. Peptide sequences detected in these plumes by mass spectrometry are encoded in the COTS genome and expressed in external tissues. The exoproteome released by aggregating COTS consists largely of signalling factors and hydrolytic enzymes, and includes an expanded and rapidly evolving set of starfish-specific ependymin-related proteins. These secreted proteins may be detected by members of a large family of olfactory-receptor-like G-protein-coupled receptors that are expressed externally, sometimes in a sex-specific manner. This study provides insights into COTS-specific communication that may guide the generation of peptide mimetics for use on reefs with COTS outbreaks.

Prolactin and the evolution of male pregnancy.

Prolactin (PRL) is a multifunctional hormone of broad physiological importance, and is involved in many aspects of fish reproduction, including the regulation of live birth (viviparity) and both male and female parental care. Previous research suggests that PRL also plays an important reproductive role in syngnathid fishes (seahorses, pipefish and seadragons), a group with a highly derived reproductive strategy, male pregnancy - how the PRL axis has come to be co-opted for male pregnancy remains unclear. We investigated the molecular evolution and expression of the genes for prolactin and its receptor (PRLR) in an evolutionarily diverse sampling of syngnathid fishes to explore how the co-option of PRL for male pregnancy has impacted its evolution, and to clarify whether the PRL axis is also involved in regulating reproductive function in species with more rudimentary forms of male pregnancy. In contrast to the majority of teleost fishes, all syngnathid fishes tested carry single copies of PRL and PRLR that cluster genetically within the PRL1 and PRLRa lineages of teleosts, respectively. PRL1 gene expression in seahorses and pipefish is restricted to the pituitary, while PRLRa is expressed in all tissues, including the brood pouch of species with both rudimentary and complex brooding structures. Pituitary PRL1 expression remains stable throughout pregnancy, but PRLRa expression is specifically upregulated in the male brood pouch during pregnancy, consistent with the higher affinity of pouch tissues for PRL hormone during embryonic incubation. Finally, immunohistochemistry of brood pouch tissues reveals that both PRL1 protein and PRLRa and Na+/K+ ATPase-positive cells line the inner pouch epithelium, suggesting that pituitary-derived PRL1 may be involved in brood pouch osmoregulation during pregnancy. Our data provide a unique molecular perspective on the evolution and expression of prolactin and its receptor during male pregnancy, and provide the foundation for further manipulative experiments exploring the role of PRL in this unique form of reproduction.

Standardization and validation of ATP luminometry as a diagnostic tool to assess the cleanliness of feeding equipment in preweaning calves.

The objective of this cross-sectional study was to standardize a reliable and repeatable swabbing technique using ATP luminometry (light emission proportional to the amount of ATP with result provided in relative light units [RLU]) to describe the cleanliness of various feeding equipment used for preweaning calves in dairy farms. A total of 7 Québec commercial dairy herds were selected conveniently. Following visual hygiene scoring, the cleanliness of every available piece of feeding equipment was assessed using direct surface swabbing for buckets and nipples with Hygiena UltraSnap swabs. A liquid rinsing technique was used for esophageal feeders, bottles, and automatic milk feeders (AMF) with UltraSnap, AquaSnap, and MicroSnap swabs. To validate direct swabbing technique of buckets, a stage within and between operators was realized, as well as a conventional bacterial culture. A total of 519 swab samples were obtained from 201 pieces of equipment. The median (interquartile range) contamination in RLU for a bottle, esophageal feeder, AMF, bucket and nipple was 2 (1;6), 2 (0;12), 52 (19;269), 886 (128;7,230) and 899 (142;6,928), respectively. The direct swabbing technique, which consists in swabbing directly the surface of an equipment, showed excellent correlation for intrarater reliability (intraclass correlation (ICC) = 0.93; 95% CI: 0.88-0.96). The interoperator (2 sessions with 3 different operators) reliability also showed high correlation (ICC = 0.88; 95% CI: 0.78-0.94 for the first session, and ICC = 0.89; 95% CI: 0.79-0.95 for the second session). Luminometer values were positively associated with the visual score of esophageal feeders, AMF and buckets. A positive correlation between bacterial culture and direct swabbing of buckets was also found for the UltraSnap (rs = 0.653; 95% CI: 0.283-0.873; P = 0.0003) and MicroSnap (rs = 0.569, 95% CI: 0.309-0.765; P = 0.002). This study describes a standardized and practical on-farm swabbing technique for assessing the hygienic status of feeding equipment by luminometry, which can be integrated in the investigation of preweaning dairy calves problems.

Hygiene management practices and adenosine triphosphate luminometry of feeding equipment in preweaning calves on dairy farms in Quebec, Canada.

The objective of this study was to describe the cleaning practices currently used for preweaning calves on dairy farms in Quebec, Canada. In addition, contamination of feeding equipment for preweaning calves was described using ATP (expressed as relative light units, RLU), visual assessment, and bacteriological analysis. A questionnaire was administered on 50 commercial dairy farms in Quebec, Canada, regarding the self-reported cleaning protocol used for feeding equipment of preweaning calves. During the visit, a visual score was given to the feeding equipment available at the farm. Afterward, ATP luminometry measurements were obtained using Hygiene UltraSnap and MicroSnap swabs (Hygiene, Camarillo, CA), and the liquid rinsing technique for buckets, nipples, bottles, esophageal tube feeders (ET), the tube of automatic milk feeders (AMF), water samples, and milk replacer. An additional direct swabbing technique was performed on buckets and nipples. The fluid retrieved from the liquid rinsing technique was also used to determine the total bacterial count (TBC) and total coliform count. Based on the bacteriological analysis, optimal RLU cutoff values to determine contamination were obtained. The median (interquartile range) luminometer measurements using the UltraSnap and direct technique for buckets and nipples were 2,082 (348-7,410) and 3,462 (462-7,518) RLU, respectively; and, using the liquid technique for bottles, ET, AMF, water, and milk replacer were 43 (4-974), 15 (4-121), 301 (137-1,323), 190 (71-358), and 94 (38-218) RLU, respectively. Overall, for all equipment and both techniques used, higher RLU values were seen in UltraSnap samples compared with MicroSnap samples. Additionally, for buckets and nipples, higher RLU values were obtained for the direct swabbing method compared with the liquid sampling method for both swabs used. No differences in the level of contamination were seen between the different feeding equipment used within a farm. Overall, a higher correlation with bacteriological results was noticed for ATP luminometry compared with the visual score, with a high correlation for nipples and bottles using the UltraSnap and liquid technique. Based on the classification of "contaminated" (TBC ≥100,000 cfu/mL) or "not contaminated" (TBC <100,000 cfu/mL), optimal ATP luminometer cutoff values for buckets, nipples, bottles, AMF, water, and milk replacer were 798, 388, 469, 282, 1,432, and 93 RLU, respectively. No clear association was found between ATP measurements and the self-reported cleaning protocol. This study gave new insights into the current cleaning procedures and contamination of feeding equipment for preweaning calves on dairy farms in Quebec. In addition, ATP luminometry cutoff values could help benchmark farms regarding cleaning practices and provide customized advice, improving the overall hygiene management, and thus the health, of preweaning calves on dairy farms.

Unphosphorylated Form of the PAQosome Core Subunit RPAP3 Binds Ribosomal Preassembly Complexes to Modulate Ribosome Biogenesis.

The PAQosome (particle for arrangement of quaternary structure) is a 12-subunit HSP90 co-chaperone involved in the biogenesis of several human protein complexes. Two mechanisms of client selection have previously been identified, namely, the selective recruitment of specific adaptors and the differential use of homologous core subunits. Here, we describe a third client selection mechanism by showing that RPAP3, one of the core PAQosome subunits, is phosphorylated at several Ser residues in HEK293 cells. Affinity purification coupled with mass spectrometry (AP-MS) using the expression of tagged RPAP3 with single phospho-null mutations at Ser116, Ser119, or Ser121 reveals binding of the unphosphorylated form to several proteins involved in ribosome biogenesis. In vitro phosphorylation assays indicate that the kinase CK2 phosphorylates these RPAP3 residues. This finding is supported by data showing that pharmacological inhibition of CK2 enhances the binding of RPAP3 to ribosome preassembly factors in AP-MS experiments. Moreover, the silencing of PAQosome subunits interferes with ribosomal assembly factors' interactome. Altogether, these results indicate that RPAP3 phosphate group addition/removal at specific residues modulates binding to subunits of preribosomal complexes and allows speculating that PAQosome posttranslational modification is a mechanism of client selection.

Methylscaper: an R/Shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data.

SUMMARY: Differential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we present methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin accessibility landscapes. Methylscaper implements a weighted principal component analysis that orders DNA molecules, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscaper's utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit-BGS) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets. AVAILABILITY AND IMPLEMENTATION: Methylscaper, is implemented in R (version > 4.1) and available on Bioconductor: https://bioconductor.org/packages/methylscaper/, GitHub: https://github.com/rhondabacher/methylscaper/, and Web: https://methylscaper.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Complete genome sequence of a novel potyvirus infecting Miscanthus sinensis (silver grass).

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named "Miscanthus sinensis mosaic virus" (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post-entry quarantine facility after being imported into Western Australia, Australia. The MsiMV genome is 9604 nucleotides (nt) in length, encoding a 3071-amino-acid (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to that of sorghum mosaic virus (SrMV), with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV), and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

Bi-allelic Mutations in EPRS, Encoding the Glutamyl-Prolyl-Aminoacyl-tRNA Synthetase, Cause a Hypomyelinating Leukodystrophy.

Hypomyelinating leukodystrophies are genetic disorders characterized by insufficient myelin deposition during development. They are diagnosed on the basis of both clinical and MRI features followed by genetic confirmation. Here, we report on four unrelated affected individuals with hypomyelination and bi-allelic pathogenic variants in EPRS, the gene encoding cytoplasmic glutamyl-prolyl-aminoacyl-tRNA synthetase. EPRS is a bifunctional aminoacyl-tRNA synthetase that catalyzes the aminoacylation of glutamic acid and proline tRNA species. It is a subunit of a large multisynthetase complex composed of eight aminoacyl-tRNA synthetases and its three interacting proteins. In total, five different EPRS mutations were identified. The p.Pro1115Arg variation did not affect the assembly of the multisynthetase complex (MSC) as monitored by affinity purification-mass spectrometry. However, immunoblot analyses on protein extracts from fibroblasts of the two affected individuals sharing the p.Pro1115Arg variant showed reduced EPRS amounts. EPRS activity was reduced in one affected individual's lymphoblasts and in a purified recombinant protein model. Interestingly, two other cytoplasmic aminoacyl-tRNA synthetases have previously been implicated in hypomyelinating leukodystrophies bearing clinical and radiological similarities to those in the individuals we studied. We therefore hypothesized that leukodystrophies caused by mutations in genes encoding cytoplasmic aminoacyl-tRNA synthetases share a common underlying mechanism, such as reduced protein availability, abnormal assembly of the multisynthetase complex, and/or abnormal aminoacylation, all resulting in reduced translation capacity and insufficient myelin deposition in the developing brain.

Associations between markers of mammary adipose tissue dysfunction and breast cancer prognostic factors.

BACKGROUND: Obesity fosters worse clinical outcomes in both premenopausal and postmenopausal women with breast cancer. Emerging evidence suggests that an android body fat distribution in particular is deleterious for breast cancer prognosis. The extent of adipose tissue dysfunction, especially how it relates to breast cancer prognostic factors and anthropometric measurements, has not been fully investigated. OBJECTIVE: Our objective was to examine if markers of adipose tissue dysfunction, such as hypertrophy and macrophage accumulation, are relevant for the pathophysiology of breast cancer and its associated prognostic factors in a well-characterised cohort of women with breast cancer who did not receive treatment before surgery. METHODS: A consecutive series of 164 women with breast cancer provided breast adipose tissue sample. Multivariate generalised linear models were used to test associations of anthropometric indices and prognostic factors with markers of adipose tissue dysfunction. RESULTS: We found associations of breast adipocyte size and macrophage infiltration (number of CD68+ cells/100 adipocytes) with adiposity, particularly a strong association between breast adipocyte size and central obesity, independent of total adiposity, age and menopausal status (ßadj = 0.87; p = 0.0001). We also identified relationships of adipocyte hypertrophy and macrophage infiltration with prognostic factors, such as cancer stage and tumour grade (p < 0.05). RNA expression of pro-inflammatory cytokines (IL6, TNF) and leptin was also increased as a function of adipocyte size and CD86+/CD11c+ macrophage number/100 adipocytes (p < 0.05). CONCLUSIONS: Our findings support the model of dysfunctional adipose tissue in obesity-associated breast cancer.

Upstream ORF-Encoded ASDURF Is a Novel Prefoldin-like Subunit of the PAQosome.

The PAQosome is an 11-subunit chaperone involved in the biogenesis of several human protein complexes. We show that ASDURF, a recently discovered upstream open reading frame (uORF) in the 5' UTR of ASNSD1 mRNA, encodes the 12th subunit of the PAQosome. ASDURF displays significant structural homology to ß-prefoldins and assembles with the five known subunits of the prefoldin-like module of the PAQosome to form a heterohexameric prefoldin-like complex. A model of the PAQosome prefoldin-like module is presented. The data presented here provide an example of a eukaryotic uORF-encoded polypeptide whose function is not limited to cis-acting translational regulation of downstream coding sequence and highlights the importance of including alternative ORF products in proteomic studies.

Molecular patterns in salivary duct carcinoma identify prognostic subgroups.

Salivary duct carcinoma (SDCa) is a rare cancer with high rate of metastases and poor survival despite aggressive multimodality treatment. This study analyzes the genetic changes in SDCa, their impact on cancer pathways, and evaluates whether molecular patterns can identify subgroups with distinct clinical characteristics and outcome. Clinicopathologic details and tissue samples from 66 patients (48 males, 18 females) treated between 1995 and 2018 were obtained from multiple institutions. Androgen receptor (AR) was assessed by immunohistochemistry, and the Illumina TruSight 170 gene panel was used for DNA sequencing. Male gender, lympho-vascular invasion, lymph node metastasis, and smoking were significant predictors of disease-free survival. AR was present in 79%. Frequently encountered alterations were mutations in TP53 (51%), PIK3CA (32%) and HRAS (22%), as well as amplifications of CDK4/6 (22%), ERBB2 (21%), MYC (16%), and deletions of CDKN2A (13%). TP53 mutation and MYC amplifications were associated with decreased disease-free survival. Analysis of cancer pathways revealed that the PI3K pathway was most commonly affected. Alterations in the cell cycle pathway were associated with impaired disease-free survival (HR 2.6, P = 0.038). Three subgroups based on AR and ERBB2 status were identified, which featured distinct molecular patterns and outcome. Among AR positive SDCa, HRAS mutations were restricted to AR positive tumors without ERBB2 amplification and HRAS mutations featured high co-occurrence with PIK3CA alterations, which seems specific to SDCa. AR negative SDCa were associated with poor disease-free survival in multivariate analysis (HR 4.5, P = 0.010) and none of these tumors exhibited ERBB2 amplification or HRAS mutations. AR and ERBB2 status in SDCa thus classifies tumors with distinct molecular profiles relevant to future targeted therapy. Furthermore, clinical factors such as smoking and molecular features such as MYC amplification may serve as markers of poor prognosis of SDCa.

Ser-Phosphorylation of PCSK9 (Proprotein Convertase Subtilisin-Kexin 9) by Fam20C (Family With Sequence Similarity 20, Member C) Kinase Enhances Its Ability to Degrade the LDLR (Low-Density Lipoprotein Receptor).

OBJECTIVE: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9. In situ hybridization of Fam20C mRNA during development and in adult mice revealed a wide tissue distribution, including liver, but not small intestine. Here, we show that Fam20C phosphorylates PCSK9 at Serines 47, 666, 668, and 688. In hepatocytes, phosphorylation enhances PCSK9 secretion and maximizes its induced degradation of the LDLR via the extracellular and intracellular pathways. Replacing any of the 4 Ser by the phosphomimetic Glu or Asp enhanced PCSK9 activity only when the other sites are phosphorylated, whereas Ala substitutions reduced it, as evidenced by Western blotting, Elisa, and LDLR-immunolabeling. This newly uncovered PCSK9/LDLR regulation mechanism refines our understanding of the implication of global PCSK9 phosphorylation in the modulation of LDL-cholesterol and rationalizes the consequence of natural mutations, for example, S668R and E670G. Finally, the relationship of Ser-phosphorylation to the implication of PCSK9 in regulating LDL-cholesterol in the neurological Fragile X-syndrome disorder was investigated. CONCLUSIONS: Ser-phosphorylation of PCSK9 maximizes both its secretion and activity on the LDLR. Mass spectrometric approaches to measure such modifications were developed and applied to quantify the levels of bioactive PCSK9 in human plasma under normal and pathological conditions.

Comprehensive Mutational and Phenotypic Characterization of New Metastatic Cutaneous Squamous Cell Carcinoma Cell Lines Reveal Novel Drug Susceptibilities.

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer. Most patients who develop metastases (2-5%) present with advanced disease that requires a combination of radical surgery and adjuvant radiation therapy. There are few effective therapies for refractory disease. In this study, we describe novel patient-derived cell lines from cSCC metastases of the head and neck (designated UW-CSCC1 and UW-CSCC2). The cell lines genotypically and phenotypically resembled the original patient tumor and were tumorogenic in mice. Differences in cancer-related gene expression between the tumor and cell lines after various culturing conditions could be largely reversed by xenografting and reculturing. The novel drug susceptibilities of UW-CSCC1 and an irradiated subclone UW-CSCC1-R to drugs targeting cell cycle, PI3K/AKT/mTOR, and DNA damage pathways were observed using high-throughput anti-cancer and kinase-inhibitor compound libraries, which correlate with either copy number variations, targetable mutations and/or the upregulation of gene expression. A secondary screen of top hits in all three cell lines including PIK3CA-targeting drugs supports the utility of targeting the PI3K/AKT/mTOR pathway in this disease. UW-CSCC cell lines are thus useful preclinical models for determining targetable pathways and candidate therapeutics.

Characterization and visualization of the liposecretion process taking place during ceiling culture of human mature adipocytes.

OBJECTIVE: To investigate and further characterize the process of mature adipocyte dedifferentiation. Our hypothesis was that dedifferentiation does not involve mitosis but rather a phenomenon of liposecretion. METHODS: Mature adipocytes were isolated by collagenase digestion of human adipose tissue samples. Ceiling cultures were established using our six-well plate model. Cells were treated with cytosine ß-d-arabinofuranoside (AraC) or vincristine (VCR), two agents blocking cell division, and were compared with vehicle. Liposecretion events were visualized by time-lapse microscopy, with and without AraC in adipocytes transducted with a baculovirus. Microscopic analyses were performed after labeling phosphorylated histone 3 and cyclin B1 in ceiling cultures. RESULTS: Treatment with AraC almost entirely prevented the formation of fibroblasts up to 12 days of ceiling culture. Similar results were obtained with VCR. The antimitotic effectiveness of the treatment was confirmed in fibroblast cultures from the adipose tissue stromal-vascular fraction by proliferation assays and colony-forming unit experiments. Using time-lapse microscopy, we visualized liposecretion events in which a large lipid droplet was rapidly secreted from isolated mature adipocytes. The same phenomenon was observed with AraC. This was observed in conjunction with histone 3 phosphorylation and cyclin B1 segregation to the nucleus. CONCLUSION: Our results support the notion that dedifferentiation involves rapid secretion of the lipid droplet by the adipocytes with concomitant generation of fibroblast-like cells that subsequently proliferate to generate the dedifferentiated adipocyte population during ceiling culture. The presence of mitotic markers suggests that this process involves cell cycle progression, although cell division does not occur.

Comparison between the enzymatic activity, structure and substrate binding of mouse and human lecithin retinol acyltransferase.

Lecithin retinol acyltransferase (LRAT) is involved in the visual cycle where it catalyzes the formation of all-trans retinyl ester. The mouse animal model has been widely used to study LRAT. Primary sequence alignment shows 80% identity and 90% similarity between human and mouse LRAT. However, human LRAT has a proline at position 173 (hLRAT (P173)) while an arginine can be found at this position for the mouse protein (mLRAT (R173)). Moreover, residue 173 is important for the human protein since a substitution mutation of this residue to a leucine (P173L-hLRAT) caused night blindness in a patient. The present study was thus undertaken to determine whether mouse and human LRAT have a similar enzymatic activity, structure and substrate binding affinity using a truncated form of LRAT (tLRAT). The enzymatic activity and binding affinity to the substrate, all-trans retinol, of mtLRAT (R173) were found to be 2.7- and 3.9-fold lower, respectively, than that of htLRAT (P173). Moreover, the enzymatic activity of P173L-htLRAT is 6.3-fold lower compared to that of htLRAT (P173). Furthermore, a significant difference was observed between the intrinsic fluorescence emission as well as between the circular dichroism spectra of mtLRAT (R173) and htLRAT (P173). In addition, mtLRAT proteins are less thermostable than htLRAT proteins, which suggests that structural differences exist between the mouse and human proteins. Altogether, these data strongly suggest that the much lower catalytic activity of mtLRAT (R173) compared to that of htLRAT (P173) mostly results from differences between their structure, predominantly revealed by their dissimilar thermal stability, as well as their efficiency to bind all-trans retinol. Therefore, conclusions regarding the behavior of human LRAT based on measurements performed with mouse LRAT must be made with caution. Also, the much lower enzymatic activity of P173L-htLRAT compared to that of htLRAT (P173) might explain the night blindness of a patient carrying this mutation.