Aligning tumor mutational burden (TMB) quantification across diagnostic platforms: phase II of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. MATERIALS AND METHODS: Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. RESULTS: Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. CONCLUSIONS: Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Alternatives to aluminum gates for silicon quantum devices: defects and strain.

Gate-defined quantum dots (QD) benefit from the use of small grain size metals for gate materials because it aids in shrinking the device dimensions. However, it is not clear what differences arise with respect to process-induced defect densities and inhomogeneous strain. Here, we present measurements of fixed charge, Q f , interface trap density, D it , the intrinsic film stress, σ, and the coefficient of thermal expansion, α as a function of forming gas anneal temperature for Al, Ti/Pd, and Ti/Pt gates. We show D it is minimal at an anneal temperature of 350 °C for all materials but Ti/Pd and Ti/Pt have higher Q f and D it compared to Al. In addition, σ and α increase with anneal temperature for all three metals with α larger than the bulk value. These results indicate that there is a tradeoff between minimizing defects and minimizing the impact of strain in quantum device fabrication.

Reduction of charge offset drift using plasma oxidized aluminum in SETs.

Aluminum oxide ([Formula: see text])-based single-electron transistors (SETs) fabricated in ultra-high vacuum (UHV) chambers using in situ plasma oxidation show excellent stabilities over more than a week, enabling applications as tunnel barriers, capacitor dielectrics or gate insulators in close proximity to qubit devices. Historically, [Formula: see text]-based SETs exhibit time instabilities due to charge defect rearrangements and defects in [Formula: see text] often dominate the loss mechanisms in superconducting quantum computation. To characterize the charge offset stability of our [Formula: see text]-based devices, we fabricate SETs with sub-1 e charge sensitivity and utilize charge offset drift measurements (measuring voltage shifts in the SET control curve). The charge offset drift ([Formula: see text]) measured from the plasma oxidized [Formula: see text] SETs in this work is remarkably reduced (best [Formula: see text] over [Formula: see text] days and no observation of [Formula: see text] exceeding [Formula: see text]), compared to the results of conventionally fabricated [Formula: see text] tunnel barriers in previous studies (best [Formula: see text] over [Formula: see text] days and most [Formula: see text] within one day). We attribute this improvement primarily to using plasma oxidation, which forms the tunnel barrier with fewer two-level system (TLS) defects, and secondarily to fabricating the devices entirely within a UHV system.

Postpartum uterine involution in sheep: histoarchitecture and changes in endometrial gene expression.

After parturition, the uterus undergoes marked remodelling during involution; however, little is known of the hormonal, cellular and molecular mechanisms that regulate this process. The working hypothesis used in this study is that return of the ovine uterus to a non-pregnant state involves termination of a hormonal servomechanism that regulates endometrial gland morphogenesis and function during pregnancy. Suffolk ewes were ovariohysterectomized on postpartum days 1, 7, 14 or 28. Serum concentrations of oestradiol were high at parturition, declined to postpartum day 4, peaked on postpartum day 6, and then declined and remained low thereafter. Progesterone was undetectable in plasma from ewes post partum. Uterine wet mass and horn length decreased after postpartum day 1, but ovarian mass did not change. Residual placental cotyledons were present in the maternal caruncles on postpartum days 1 and 7 and were extruded by postpartum day 14 as plaques that were resorbed by postpartum day 28. The width of the total endometrium, stratum compactum, stratum spongiosum and myometrium, as well as endometrial gland density, decreased after parturition. Most apoptotic cells in the involuting uterus were large, vacuolated and located between the endometrial glandular epithelial cells on postpartum days 1 and 7. Immunofluorescence analyses identified both T and B cells within the glandular epithelium on postpartum day 1. Cell proliferation was detected in the luminal epithelium and glandular epithelium on postpartum days 1 and 7. On postpartum day 1, expression of oestrogen receptor alpha (ERalpha) was not detected in luminal epithelium and was low in glandular epithelium, but ERalpha was present in epithelia thereafter. Progesterone receptor (PR) protein was not detected in endometrial epithelia on postpartum day 1, but was detected in the glandular epithelium thereafter. Between postpartum days 1 and 7, ERalpha and PR protein increased substantially in the endometrial glandular epithelium. On postpartum days 1-28, abundant expression of oxytocin receptor mRNA was detected in endometrial luminal epithelium and superficial to the middle glandular epithelium. Prolactin receptor (PRLR) mRNA was detected in glandular epithelium on all postpartum days, whereas mRNA for uterine milk protein (UTMP), an index of secretory capacity of glandular epithelium, was present only on postpartum day 1. Collectively, these results indicate that uterine involution in ewes involves remodelling of both caruncular and intercaruncular areas of the uterine wall and termination of differentiated uterine gland functions characteristic of pregnancy.

Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus.

Interferon tau (IFNtau) is the signal for maternal recognition of pregnancy in ruminants. The positive effects of IFNtau on IFN-stimulated gene (ISG) expression are mediated by ISG factor 3 (ISGF3), which is composed of signal transducer and activator of transcription (Stat) 1, Stat 2, and IFN regulatory factor-9 (IRF-9), and by gamma-activated factor (GAF), which is a Stat 1 homodimer. Induction of ISGs, such as ISG17 and 2',5'-oligoadenylate synthetase, by IFNtau during pregnancy is limited to the endometrial stroma (S) and glandular epithelium (GE) of the ovine uterus. The IRF-2, a potent transcriptional repressor of ISG expression, is expressed in the luminal epithelium (LE). This study determined effects of the estrous cycle, pregnancy, and IFNtau on expression of Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 genes in the ovine endometrium. In cyclic ewes, Stat 1, Stat 2, IRF-1, and IRF-9 mRNA and protein were detected at low levels in the S and GE. During pregnancy, expression of these genes increased only in the S and GE. Expression of IRF-2 was detected only in the LE and superficial GE (sGE) of both cyclic and pregnant ewes. In cyclic ewes, intrauterine administration of IFNtau stimulated Stat 1, Stat 2, IRF-9, and IRF-1 expression in the endometrium. Ovine IRF-2 repressed transcriptional activity driven by IFN-stimulated response elements that bind ISGF3, but not by gamma-activation sequences that bind GAF. These results suggest that IRF-2 in the LE and sGE restricts IFNtau induction of ISGs to the S and GE. In the S and GE, IFNtau hyperactivation of ISG expression likely involves formation and actions of the transcription factors ISGF3 and, perhaps, IRF-1.

Biological effects of embedded depleted uranium (DU): summary of armed forces radiobiology research institute research.

The Persian Gulf War resulted in injuries of US Coalition personnel by fragments of depleted uranium (DU). Fragments not immediately threatening the health of the individuals were allowed to remain in place, based on long-standing treatment protocols designed for other kinds of metal shrapnel injuries. However, questions were soon raised as to whether this approach is appropriate for a metal with the unique radiological and toxicological properties of DU. The Armed Forces Radiobiology Research Institute (AFRRI) is investigating health effects of embedded fragments of DU to determine whether current surgical fragment removal policies remain appropriate for this metal. These studies employ rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate uranium from implanted DU fragments distributed to tissues far-removed from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in the kidney that were nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed. However, results suggest the need for further studies of long-term health impact, since DU was found to be mutagenic, and it transformed human osteoblast cells to a tumorigenic phenotype. It also altered neurophysiological parameters in rat hippocampus, crossed the placental barrier, and entered fetal tissue. This report summarizes AFRRI's depleted uranium research to date.

Interferon-tau (IFNtau) regulation of IFN-stimulated gene expression in cell lines lacking specific IFN-signaling components.

Interferon-tau (IFNtau) is a unique type I IFN secreted by the ruminant conceptus that acts in a paracrine manner on the endometrial epithelium to signal pregnancy recognition. In the ovine endometrium, IFNtau suppresses estrogen receptor alpha and oxytocin receptor gene expression, but increases or induces expression of IFN-simulated genes (ISGs), including signal transducer and activator of transcription-1 (STAT1), STAT2, ISG factor-3gamma (ISGF3gamma)/p48/IFN regulatory factor-9, and 2',5'-oligoadenylate synthetase (OAS). Human fibroblast cell lines lacking specific IFN signaling components were employed to determine the roles of STAT1, STAT2, and ISGF3gamma in the effects of IFNtau on ISG protein expression. Results indicated that STAT1alpha or STAT1beta is required for IFNtau effects on STAT2, ISGF3gamma, and OAS (40/46, 69/71, and 100 kDa). STAT2 is required for effects on STAT1, ISGF3gamma, and all OAS forms. ISGF3gamma is required for effects of IFNtau on STAT2 and 40/46- and 69/71-kDa OAS and plays a role in the effects of IFNtau on 100-kDa OAS and STAT1. Mutation of Tyr(701), but not Ser(727), of STAT1 abolished the effects of IFNtau on ISG expression. Mutation of the SH2 domain of STAT1 abolished the effects of IFNtau on all ISGs and reduced increases in 100-kDa OAS. These data illustrate the importance of transcription factors composed of STAT1, STAT2, and ISGF3gamma in the signaling pathway mediating the effects of IFNtau on ISG expression.

Effects of the estrous cycle, pregnancy, and interferon tau on 2',5'-oligoadenylate synthetase expression in the ovine uterus.

The enzymes which comprise the 2',5'-oligoadenylate synthetase (OAS) family are interferon (IFN) stimulated genes which regulate ribonuclease L antiviral responses and may play additional roles in control of cellular growth and differentiation. This study characterized OAS expression in the endometrium of cyclic and pregnant ewes as well as determined effects of IFNtau and progesterone on OAS expression in cyclic or ovariectomized ewes and in endometrial epithelial and stromal cell lines. In cyclic ewes, low levels of OAS protein were detected in the endometrial stroma (S) and glandular epithelium (GE). In early pregnant ewes, OAS expression increased in the S and GE on Day 15. OAS expression in the lumenal epithelium (LE) was not detected in uteri from either cyclic or pregnant ewes. Intrauterine administration of IFNtau stimulated OAS expression in the S and GE, and this effect of IFNtau was dependent on progesterone. Ovine endometrial LE, GE, and S cell lines responded to IFNtau with induction of OAS proteins. In all three cell lines, the 40/46-kDa OAS forms were induced by IFNtau, whereas the 100-kDa OAS form appeared to be constitutively expressed and not affected by IFNtau. The 69/71-kDa OAS forms were induced by IFNtau in the S and GE cell lines, but not in the LE. Collectively, these results indicate that OAS expression in the endometrial S and GE of the early pregnant ovine uterus is directly regulated by IFNtau from conceptus and requires the presence of progesterone.

Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells.

Interferon-tau (IFNtau), a type I IFN produced by sheep conceptus trophectoderm, is the signal for maternal recognition of pregnancy. Although it is clear that IFNtau suppresses transcription of the estrogen receptor alpha and oxytocin receptor genes and induces expression of various IFN-stimulated genes within the endometrial epithelium, little is known of the signal transduction pathway activated by the hormone. This study determined the effects of IFNtau on signal transducer and activator of transcription (STAT) activation, expression, DNA binding, and transcriptional activation using an ovine endometrial epithelial cell line. IFNtau induced persistent tyrosine phosphorylation and nuclear translocation of STAT1 and -2 (10 min to 48 h), but transient phosphorylation and nuclear translocation of STAT3, -5a/b, and -6 (10 to <60 min). IFNtau increased expression of STAT1 and -2, but not STAT3, -5a/b, and -6. IFN-stimulated gene factor-3 and STAT1 homodimers formed and bound an IFN-stimulated response element (ISRE) and gamma-activated sequence (GAS) element, respectively. IFNtau increased transcription of GAS-driven promoters at 3 h, but suppressed their activity at 24 h. In contrast, the activity of an ISRE-driven promoter was increased at 3 and 24 h. These results indicate that IFNtau activates multiple STATs and has differential effects on ISRE- and GAS-driven gene transcription.

Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin.

Effects of age on uterine histoarchitecture, cell proliferation, and hormone receptor expression were determined for neonatal ewe lambs from birth (Postnatal Day [PND] 0) to PND 56. Uteri were histologically evaluated and proliferating cell nuclear antigen (PCNA), estrogen receptor alpha (ER-alpha), progesterone receptor (PR), and prolactin receptor (PRL-R) expression were characterized by in situ hybridization (ISH), immunohistochemistry, or both. The most striking feature of neonatal uterine development was the genesis and development of glands in the intercaruncular areas of endometrium. After birth, endometrial glandular epithelium (GE) budded and differentiated into the underlying stroma from the luminal epithelium (LE) between PNDs 1 and 7. Between PNDs 14 and 56, extensive coiling and branching morphogenesis of nascent endometrial glands occurred. By PND 56, the uterine wall appeared to be histoarchitecturally mature. At birth, nuclear PCNA protein was strongly detected in LE. Between PNDs 7 and 56, high levels of PCNA, ER-alpha, and PR gene expression were detected in both nascent and developing GE. Higher levels of PCNA and ER-alpha expression were detected in GE at the tips of developing glands as well as in the surrounding stroma. Progesterone was below detectable limits in serum. Serum estradiol-17beta levels were high on PND 1, increased from PNDs 14 to 28, and declined from PND 42 to PND 56. Serum PRL levels increased from PNDs 1 to 14 and declined thereafter. Using ISH and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, expression of mRNAs for short and long forms of the ovine PRL-R were first detected in nascent GE on PND 7 and increased between PNDs 7 and 56 in proliferating and differentiating GE. These results indicate that 1) uterine gland genesis is initiated between PNDs 1 and 7 after birth and is essentially completed by PND 56; 2) neonatal uterine morphogenesis involves temporal and spatial alterations in cell proliferation and ER-alpha, PR, and PRL-R gene expression; 3) PRL-R expression is a unique marker of GE differentiation and proliferation; and 4) serum estradiol-17beta and PRL levels increase during the onset of GE tubular branching morphogenesis. Results support the hypothesis that neonatal ovine uterine development involves epithelial PRL-R and ER-alpha activation to stimulate and maintain endometrial gland genesis and branching morphogenesis.

Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy.

Lactogenic hormones regulate epithelial proliferation, differentiation, and function in a variety of epitheliomesenchymal organs. During pregnancy, the ovine uterus is a potential site for endocrine and paracrine actions of lactogenic hormones in the form of pituitary prolactin (PRL) and placental lactogen (PL). These studies determined temporal and spatial alterations in PRL receptor (PRL-R) and expression of uterine milk proteins (UTMP), a marker of endometrial secretory activity, in the ovine endometrium during the estrous cycle and pregnancy. Slot-blot hybridization analysis indicated that steady-state levels of endometrial PRL-R mRNA increased during pregnancy. In situ hybridization and immunohistochemical analyses indicated that PRL-R mRNA and protein were exclusively expressed in the endometrial glandular epithelium (GE). No PRL-R mRNA expression was detected in luminal epithelium, stroma, myometrium, or conceptus trophectoderm. Reverse transcription-polymerase chain reaction analyses determined that the endometrial GE expressed both long and short alternative splice forms of the ovine PRL-R gene. Slot-blot hybridization analysis indicated that steady-state levels of intercaruncular endometrial UTMP mRNA increased about 3-fold between Days 20 and 60, increased another 3-fold between Days 60 and 80, and then declined slightly to Day 120. In pregnant ewes, UTMP mRNA expression was restricted to the endometrial GE in the stratum spongiosum (sGE), increased substantially between Days 15 and 17, and, between Days 17 to 50 of gestation, was markedly higher in upper than lower sGE. After Day 50, hyperplasia of the sGE was accompanied by increased UTMP mRNA expression by all sGE. Collectively, results indicate that 1) endometrial sGE is a primary target for actions of lactogenic hormones and 2) UTMP mRNA expression is correlated with PL production by the trophectoderm and state of sGE differentiation during pregnancy. It is proposed that activation of PRL-R signal transduction pathways by PRL and PL plays a major role in endometrial GE remodeling and differentiated function during pregnancy in support of conceptus growth and development.

Subclavian and axillary involvement in temporal arteritis and polymyalgia rheumatica.

PURPOSE: We describe 10 female patients with temporal arteritis (TA) and/or polymyalgia rheumatica (PMR) who presented with upper-extremity ischemia. PATIENTS, METHODS, AND RESULTS: Arm claudication or Raynaud's phenomenon was the initial manifestation of the disease in four cases, appeared with classical symptoms in one case, or occurred during decreasing corticosteroid therapy in five cases. Temporal artery biopsy was performed in nine patients and showed typical giant-cell granulomatous arteritis in seven cases. Angiograms in all cases showed multiple bilateral smooth stenoses, or obliterations of postvertebral subclavian and/or axillary arteries, or both. Symptoms always improved with corticosteroid treatment and none of the patients required reconstructive surgery, although angiography performed after stabilization did not show revascularization of occluded vessels. CONCLUSION: We conclude that large-artery involvement in TA and PMR affects most commonly the subclavian and axillary arteries, with a female predominance comparable to that in Takayasu's arteritis. Both these disorders should be considered in elderly women with occlusive disease of the upper extremities. Although response to steroid therapy was sufficient in our series to avoid surgery, we believe it is preferable to recognize large-artery involvement as early as possible and recommend performance of ultrasonic Doppler examination when any sign of oncoming ischemia or stenosis is observed.