Proline biosynthesis regulates proline transport in Staphylococcus aureus.

Staphylococcus aureus is metabolically diverse with the ability to rapidly adapt to a vast array of nutrient sources. This allows the pathogen to colonize a variety of niches in the host. For instance, S. aureus is the leading cause of skin and soft tissue infections, a niche that has been shown to become glucose-depleted over the course of an infection. Previous studies have shown that in niches where glucose is deficient, S. aureus utilizes peptides and free amino acids as nutrient sources. Primarily, these amino acids include glutamate and amino acids that can serve as substrates for glutamate synthesis. While arginine and histidine serve as substrates in glutamate synthesis, proline is the primary source of glutamate. Indeed, S. aureus utilizes proline as a secondary carbon source only when glucose is absent, and it can be synthesized from arginine or acquired via proline transporters from its environment. Although S. aureus encodes two putative pathways for proline biosynthesis, it has been shown that pyrroline-5-carboxylate reductase (encoded by proC) is the sole proline biosynthetic pathway in S. aureus. Studies from our laboratory have revealed that despite encoding five putative proline transporters (B7H15_03660, opuC, opuD, proP, putP), only two of the transporters, PutP and B7H15_03660 are responsible for a majority of proline transport under the laboratory conditions tested. Surprisingly, when we introduced the proC mutation into the B7H15_03660 putP double mutant, we observed proline-dependent growth, even though the primary proline transporters and proline biosynthetic pathway were knocked-out. In contrast, a transporter null ΔproC strain was unable to grow. These data suggest that inhibiting proline biosynthesis alters proline transport, and therefore one or more of the additional transporters, OpuC, OpuD, and/or ProP, are activated under these conditions. After introducing opuC, opuD, and/or proP mutations into the Δ03660 ΔputP ΔproC strain, we found that both OpuC and ProP are important for proline transport. Additionally, we observed proline-dependent growth in a proline transporter null ΔproC strain when high amounts of exogenous proline are added to the media. This growth appears to be due to an acquired mutation and will be studied more in the future. Overall these studies have revealed that proline transport is tightly linked to proline biosynthesis.https://digitalcommons.unmc.edu/surp2021/1021/thumbnail.jp

Primary PEComa of the bladder treated with primary excision and adjuvant interferon-alpha immunotherapy: a case report

BACKGROUND: Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal neoplasms of uncertain malignant potential, which have in common the co-expression of muscle and melanocytic immunohistochemical markers. CASE PRESENTATION: A 48-year-old man presented with dysuria, passage of urinary sediment and lower abdominal discomfort. A three centimeter mass was identified by cystoscopy in the posterior midline of the bladder. Computerized tomography suggested an enterovesical fistula. The patient underwent laparotomy, partial cystectomy and partial small bowel resection. Pathological examination revealed PEComa of the bladder. The patient underwent adjuvant interferon-α immunotherapy. Subsequent follow-up procedures, including cystoscopy and imaging, have not revealed evidence of recurrence. The patient is clinically free of disease 48 months after surgery. CONCLUSION: This case represents the second documented PEComa of bladder and demonstrates that adjuvant therapies, including anti-angiogenic and immunotherapy, may be considered for patients with locally advanced or metastatic genitourinary PEComa

The ubiquitin proteasome system in neuropathology

The ubiquitin proteasome system (UPS) orchestrates the turnover of innumerable cellular proteins. In the process of ubiquitination the small protein ubiquitin is attached to a target protein by a peptide bond. The ubiquitinated target protein is subsequently shuttled to a protease complex known as the 26S proteasome and subjected to degradative proteolysis. The UPS facilitates the turnover of proteins in several settings. It targets oxidized, mutant or misfolded proteins for general proteolytic destruction, and allows for the tightly controlled and specific destruction of proteins involved in development and differentiation, cell cycle progression, circadian rhythms, apoptosis, and other biological processes. In neuropathology, alteration of the UPS, or mutations in UPS target proteins may result in signaling abnormalities leading to the initiation or progression of tumors such as astrocytomas, hemangioblastomas, craniopharyngiomas, pituitary adenomas, and medulloblastomas. Dysregulation of the UPS may also contribute to tumor progression by perturbation of DNA replication and mitotic control mechanisms, leading to genomic instability. In neurodegenerative diseases caused by the expression of mutant proteins, the cellular accumulation of these proteins may overload the UPS, indirectly contributing to the disease process, e.g., sporadic Parkinsonism and prion diseases. In other cases, mutation of UPS components may directly cause pathological accumulation of proteins, e.g., autosomal recessive Parkinsonism and spinocerebellar ataxias. Defects or dysfunction of the UPS may also underlie cognitive disorders such as Angelman syndrome, Rett syndrome and autism, and muscle and nerve diseases, e.g., inclusion body myopathy and giant axon neuropathy. This paper describes the basic biochemical mechanisms comprising the UPS and reviews both its theoretical and proven involvement in neuropathological diseases. The potential for the UPS as a target of pharmacological therapy is also discussed

Validity and responsiveness of the Clubfoot Assessment Protocol (CAP). A methodological study

BACKGROUND: The Clubfoot Assessment Protocol (CAP) is a multi dimensional instrument designed for longitudinal follow up of the clubfoot deformity during growth. Item reliability has shown to be sufficient. In this article the CAP's validity and responsiveness is studied using the Dimeglio classification scoring as a gold standard. METHODS: Thirty-two children with 45 congenital clubfeet were assessed prospectively and consecutively at ages of new-born, one, two, four months and two years of age. For convergent/divergent construct validity the Spearman's correlation coefficients were calculated. Discriminate validity was evaluated by studying the scores in bilateral clubfeet. The floor-ceiling effects at baseline (untreated clubfeet) and at two years of age (treated clubfeet) were evaluated. Responsiveness was evaluated by using effect sizes (ES) and by calculating if significant changes (Wilcoxons signed test) had occurred between the different measurement occasions. RESULTS: High to moderate significant correlation were found between CAP mobility I and morphology and the Dimeglio scores (r(s )= 0.77 and 0.44 respectively). Low correlation was found between CAP muscle function, mobility II and motion quality and the Dimeglio scoring system (r(s )= 0.20, 0.09 and 0.06 respectively). Of 13 children with bilateral clubfeet, 11 showed different CAP mobility I scores between right and left foot at baseline (untreated) compared with 5 with the Dimeglio score. At the other assessment occasions the CAP mobility I continued to show higher discrimination ability than the Dimeglio. No floor effects and low ceiling effects were found in the untreated clubfeet for both instruments. High ceiling effects were found in the CAP for the treated children and low for the Dimeglio. Responsiveness was good. ES from untreated to treated ranged from 0.80 to 4.35 for the CAP subgroups and was 4.68 for the Dimeglio. The first four treatment months, the CAP mobility I had generally higher ES compared with the Dimeglio. CONCLUSION: The Clubfoot Assessment Protocol shows in this study good validity and responsiveness. The CAP is more responsive when severity ranges between mild – moderate to severe, while the Dimeglio focuses more on the extremes. The ability to discriminate between different mobility status of the right and left foot in bilaterally affected children in this population was higher compared with the Dimeglio score implicating a better sensitivity for the CAP

Computational prediction and experimental validation associating FABP-1 and pancreatic adenocarcinoma with diabetes

<p/> <p>Background</p> <p>Pancreatic cancer, composed principally of pancreatic adenocarcinoma (PaC), is the fourth leading cause of cancer death in the United States. PaC-associated diabetes may be a marker of early disease. We sought to identify molecules associated with PaC and PaC with diabetes (PaC-DM) using a novel translational bioinformatics approach. We identified fatty acid binding protein-1 (FABP-1) as one of several candidates. The primary aim of this pilot study was to experimentally validate the predicted association between FABP-1 with PaC and PaC with diabetes.</p> <p>Methods</p> <p>We searched public microarray measurements for genes that were specifically highly expressed in PaC. We then filtered for proteins with known involvement in diabetes. Validation of FABP-1 was performed via antibody immunohistochemistry on formalin-fixed paraffin embedded pancreatic tissue microarrays (FFPE TMA). FFPE TMA were constructed using148 cores of pancreatic tissue from 134 patients collected between 1995 and 2002 from patients who underwent pancreatic surgery. Primary analysis was performed on 21 normal and 60 pancreatic adenocarcinoma samples, stratified for diabetes. Clinical data on samples was obtained via retrospective chart review. Serial sections were cut per standard protocol. Antibody staining was graded by an experienced pathologist on a scale of 0-3. Bivariate and multivariate analyses were conducted to assess FABP-1 staining and clinical characteristics.</p> <p>Results</p> <p>Normal samples were significantly more likely to come from younger patients. PaC samples were significantly more likely to stain for FABP-1, when FABP-1 staining was considered a binary variable. Compared to normals, there was significantly increased staining in diabetic PaC samples (p = 0.004) and there was a trend towards increased staining in the non-diabetic PaC group (p = 0.07). In logistic regression modeling, FABP-1 staining was significantly associated with diagnosis of PaC (OR 8.6 95% CI 1.1-68, p = 0.04), though age was a confounder.</p> <p>Conclusions</p> <p>Compared to normal controls, there was a significant positive association between FABP-1 staining and PaC on FFPE-TMA, strengthened by the presence of diabetes. Further studies with closely phenotyped patient samples are required to understand the true relationship between FABP-1, PaC and PaC-associated diabetes. A translational bioinformatics approach has potential to identify novel disease associations and potential biomarkers in gastroenterology.</p

Immunization with Cocktail of HIV-Derived Peptides in Montanide ISA-51 Is Immunogenic, but Causes Sterile Abscesses and Unacceptable Reactogenicity

BACKGROUND: A peptide vaccine was produced containing B and T cell epitopes from the V3 and C4 Envelope domains of 4 subtype B HIV-1 isolates (MN, RF, CanO, & Ev91). The peptide mixture was formulated as an emulsion in incomplete Freund's adjuvant (IFA). METHODS: Low-risk, healthy adult subjects were enrolled in a randomized, placebo-controlled dose-escalation study, and selected using criteria specifying that 50% in each study group would be HLA-B7+. Immunizations were scheduled at 0, 1, and 6 months using a total peptide dose of 1 or 4 mg. Adaptive immune responses in16 vaccine recipients and two placebo recipients after the 2nd immunization were evaluated using neutralization assays of sera, as well as ELISpot and ICS assays of cryopreserved PBMCs to assess CD4 and CD8 T-cell responses. In addition, (51)Cr release assays were performed on fresh PBMCs following 14-day stimulation with individual vaccine peptide antigens. RESULTS: 24 subjects were enrolled; 18 completed 2 injections. The study was prematurely terminated because 4 vaccinees developed prolonged pain and sterile abscess formation at the injection site-2 after dose 1, and 2 after dose 2. Two other subjects experienced severe systemic reactions consisting of headache, chills, nausea, and myalgia. Both reactions occurred after the second 4 mg dose. The immunogenicity assessments showed that 6/8 vaccinees at each dose level had detectable MN-specific neutralizing (NT) activity, and 2/7 HLA-B7+ vaccinees had classical CD8 CTL activity detected. However, using both ELISpot and ICS, 8/16 vaccinees (5/7 HLA-B7+) and 0/2 controls had detectable vaccine-specific CD8 T-cell responses. Subjects with moderate or severe systemic or local reactions tended to have more frequent T cell responses and higher antibody responses than those with mild or no reactions. CONCLUSIONS: The severity of local responses related to the formulation of these four peptides in IFA is clinically unacceptable for continued development. Both HIV-specific antibody and T cell responses were induced and the magnitude of response correlated with the severity of local and systemic reactions. If potent adjuvants are necessary for subunit vaccines to induce broad and durable immune responses, careful, incremental clinical evaluation is warranted to minimize the risk of adverse events. TRIAL REGISTRATION: ClinicalTrials.gov NCT00000886

Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma

Therapy development for adult diffuse glioma is hindered by incomplete knowledge of somatic glioma driving alterations and suboptimal disease classification. We defined the complete set of genes associated with 1,122 diffuse grade II-III-IV gliomas from The Cancer Genome Atlas and used molecular profiles to improve disease classification, identify molecular correlations, and provide insights into the progression from low- to high-grade disease. Whole-genome sequencing data analysis determined that ATRX but not TERT promoter mutations are associated with increased telomere length. Recent advances in glioma classification based on IDH mutation and 1p/19q co-deletion status were recapitulated through analysis of DNA methylation profiles, which identified clinically relevant molecular subsets. A subtype of IDH mutant glioma was associated with DNA demethylation and poor outcome; a group of IDH-wild-type diffuse glioma showed molecular similarity to pilocytic astrocytoma and relatively favorable survival. Understanding of cohesive disease groups may aid improved clinical outcomes

APCcdh1 Mediates Degradation of the Oncogenic Rho-GEF Ect2 after Mitosis

Background: Besides regulation of actin cytoskeleton-dependent functions, Rho GTPase pathways are essential to cell cycle progression and cell division. Rho, Rac and Cdc42 regulate G1 to S phase progression and are involved in cytokinesis. RhoA GDP/GTP cycling is required for normal cytokinesis and recent reports have shown that the exchange factor Ect2 and the GTPase activating protein MgcRacGAP regulate RhoA activity during mitosis. We previously showed that the transcription factors E2F1 and CUX1 regulate expression of MgcRacGAP and Ect2 as cells enter S-phase. Methodology/Principal Findings: We now report that Ect2 is subject to proteasomal degradation after mitosis, following ubiquitination by the APC/C complex and its co-activator Cdh1. A proper nuclear localization of Ect2 is necessary for its degradation. APC-Cdh1 assembles K11-linked poly-ubiquitin chains on Ect2, depending upon a stretch of,25 amino acid residues that contain a bi-partite NLS, a conventional D-box and two TEK-like boxes. Site-directed mutagenesis of target sequences generated stabilized Ect2 proteins. Furthermore, such degradation-resistant mutants of Ect2 were found to activate RhoA and subsequent signalling pathways and are able to transform NIH3T3 cells. Conclusions/Significance: Our results identify Ect2 as a bona fide cell cycle-regulated protein and suggest that its ubiquitination-dependent degradation may play an important role in RhoA regulation at the time of mitosis. Our findings raise the possibility that the overexpression of Ect2 that has been reported in some human tumors might result not only from deregulated transcription, but also from impaired degradation

Expression of Constitutively Active CDK1 Stabilizes APC-Cdh1 Substrates and Potentiates Premature Spindle Assembly and Checkpoint Function in G1 Cells

Mitotic progression in eukaryotic cells depends upon the activation of cyclin-dependent kinase 1 (CDK1), followed by its inactivation through the anaphase-promoting complex (APC)/cyclosome-mediated degradation of M-phase cyclins. Previous work revealed that expression of a constitutively active CDK1 (CDK1AF) in HeLa cells permitted their division, but yielded G1 daughter cells that underwent premature S-phase and early mitotic events. While CDK1AF was found to impede the sustained activity of APC-Cdh1, it was unknown if this defect improperly stabilized mitotic substrates and contributed to the occurrence of these premature M phases. Here, we show that CDK1AF expression in HeLa cells improperly stabilized APC-Cdh1 substrates in G1-phase daughter cells, including mitotic kinases and the APC adaptor, Cdc20. Division of CDK1AF-expressing cells produced G1 daughters with an accelerated S-phase onset, interrupted by the formation of premature bipolar spindles capable of spindle assembly checkpoint function. Further characterization of these phenotypes induced by CDK1AF expression revealed that this early spindle formation depended upon premature CDK1 and Aurora B activities, and their inhibition induced rapid spindle disassembly. Following its normal M-phase degradation, we found that the absence of Wee1 in these prematurely cycling daughter cells permitted the endogenous CDK1 to contribute to these premature mitotic events, since expression of a non-degradable Wee1 reduced the number of cells that exhibited premature cyclin B1oscillations. Lastly, we discovered that Cdh1-ablated cells could not be forced into a premature M phase, despite cyclin B1 overexpression and proteasome inhibition. Together, these results demonstrate that expression of constitutively active CDK1AF hampers the destruction of critical APC-Cdh1 targets, and that this type of condition could prevent newly divided cells from properly maintaining a prolonged interphase state. We propose that this more subtle type of defect in activity of the APC-driven negative-feedback loop may have implications for triggering genome instability and tumorigenesis

The Onconeural Antigen cdr2 Is a Novel APC/C Target that Acts in Mitosis to Regulate C-Myc Target Genes in Mammalian Tumor Cells

Cdr2 is a tumor antigen expressed in a high percentage of breast and ovarian tumors and is the target of a naturally occurring tumor immune response in patients with paraneoplastic cerebellar degeneration, but little is known of its regulation or function in cancer cells. Here we find that cdr2 is cell cycle regulated in tumor cells with protein levels peaking in mitosis. As cells exit mitosis, cdr2 is ubiquitinated by the anaphase promoting complex/cyclosome (APC/C) and rapidly degraded by the proteasome. Previously we showed that cdr2 binds to the oncogene c-myc, and here we extend this observation to show that cdr2 and c-myc interact to synergistically regulate c-myc-dependent transcription during passage through mitosis. Loss of cdr2 leads to functional consequences for dividing cells, as they show aberrant mitotic spindle formation and impaired proliferation. Conversely, cdr2 overexpression is able to drive cell proliferation in tumors. Together, these data indicate that the onconeural antigen cdr2 acts during mitosis in cycling cells, at least in part through interactions with c-myc, to regulate a cascade of actions that may present new targeting opportunities in gynecologic cancer