Ultraviolet dosage and decontamination efficacy were widely variable across 14 UV devices after testing a dried enveloped ribonucleic acid virus surrogate for SARS-CoV-2.

Aims: The dosages and efficacy of 14 ultraviolet (UV) decontamination technologies were measured against a SARS-CoV-2 surrogate virus that was dried onto different materials for laboratory and field testing. Methods and results: A live enveloped, ribonucleic acid (RNA) virus surrogate for SARS-CoV-2 was dried on stainless steel 304 (SS304), Navy Top Coat-painted SS304 (NTC), cardboard, polyurethane, polymethyl methacrylate (PMMA), and acrylonitrile butadiene styrene (ABS) materials at > 8.0 log10 plaque-forming units (PFU) per test coupon. The coupons were then exposed to UV radiation during both laboratory and field testing. Commercial and prototype UV-emitting devices were measured for efficacy: four handheld devices, three room/surface-disinfecting machines, five air disinfection devices, and two larger custom-made machines. UV device dosages ranged from 0.01 to 729 mJ cm-2. The antiviral efficacy among the different UV devices ranged from no decontamination up to nearly achieving sterilization. Importantly, cardboard required far greater dosage than SS304. Conclusion: Enormous variability in dosage and efficacy was measured among the different UV devices. Porous materials limit the utility of UV decontamination. Significance and impact of the study: UV devices have wide variability in dosages, efficacy, hazards, and UV output over time, indicating that each UV device needs independent technical measurement and assessment for product development prior to and during use.

Hot, Humid Air Decontamination of Aircraft Confirmed That High Temperature and High Humidity Are Critical for Inactivation of Infectious, Enveloped Ribonucleic Acid (RNA) Virus.

Aims: To develop infectious (live/dead) enveloped virus test indicators and response surface methodology (RSM) models that evaluate survival of an enveloped ribonucleic acid (RNA) virus on contaminated aircraft materials after exposure to hot, humid air (HHA). Methods and Results: Enveloped RNA bacteriophage Phi6 (Φ6) was dried on wiring insulation, aircraft performance coating (APC), polypropylene, and nylon at ≥ 8 log10 plaque-forming units (PFU) test coupon-1. Only 2.4 log10 inactivation was measured on APC at 70°Celsius (°C), 5% relative humidity (RH) after 24 h. In contrast, HHA RSM models showed a 90% probability of a 7 log10 inactivation at ≥63°C, 90% RH after 1 h, and decontamination kinetics were similar across different materials. HHA decontamination of C-130 and C-17 aircraft showed >7 log10 and ≥5.9 log10 inactivation of enveloped virus on 100 and 110 test indicators, respectively, with a 1-h treatment, excluding ramp-up and ramp-down times. Conclusions: Enveloped RNA virus test indicators were successfully developed, lab tested for HHA decontamination, analyzed for RSM, and field-tested in aircraft demonstrations. Significance and Impact of the Study: The utility of HHA decontamination was demonstrated after inactivating enveloped RNA virus on aircraft with a 1-h HHA treatment within aircraft temperature and RH limits.

Cdc7p-Dbf4p regulates mitotic exit by inhibiting Polo kinase.

Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit binds Cdc7p and is essential for Cdc7p kinase activation, however, the N-terminal third of Dbf4p is dispensable for its essential replication activities. Here, we define a short N-terminal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cdc5p, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p does not inhibit Polo kinase activity, it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN), presumably by altering Polo substrate targeting. In addition, although dbf4 mutants defective for interaction with Polo transit S-phase normally, they aberrantly segregate chromosomes following nuclear misorientation. Therefore, Cdc7p-Dbf4p prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint.

The role of intrinsic efficacy in determining response to a beta2-agonist in acute severe asthma.

BACKGROUND: Current guidelines recommend repeated doses of albuterol for the emergency treatment of acute asthma. However, approximately one-third of patients show little or no initial response to this partial beta(2)-agonist. METHODS: We conducted a randomized, double-blind, proof-of-concept study to investigate whether a full beta(2)-agonist, isoproterenol, offers a therapeutic advantage in adults presenting with acute severe asthma (FEV(1)<50%) who fail to respond to an initial treatment of the partial beta(2)-agonist, albuterol. Study subjects were randomized to receive a 2-h continuous nebulization of either albuterol (7.5mg/h) (n=10, mean FEV(1)=37% predicted) or isoproterenol (7.5mg/h) (n=9, mean FEV(1)=33% predicted). Respiratory symptoms, vital signs and pulmonary function measures were collected. RESULTS: Subjects from both treatment groups had similar baseline characteristics. The percent improvements from baseline FEV(1) at 60 and 120min were significantly higher in subjects receiving isoproterenol than those receiving albuterol (44 vs. 17% and 63 vs. 24%, respectively, P<0.05). The change in symptoms measured by the modified Borg score was also significantly greater in subjects receiving isoproterenol (P<0.01). Both treatments were well tolerated, though the mean increase in pulse rate at 60 and 120min (21 vs. 1 and 23 vs. 6beats/min, respectively, P<0.05) and the mean change in serum potassium at 120min (-0.52 vs. -0.07meq/L, P<0.05) from baseline were significantly greater in the isoproterenol group. CONCLUSIONS: Our data suggest that in subjects presenting with acute severe asthma who fail to show an initial response to albuterol, the use of a beta(2)-agonist of higher intrinsic efficacy can be more effective in improving lung function and symptoms.

A Dbf4p BRCA1 C-terminal-like domain required for the response to replication fork arrest in budding yeast.

Dbf4p is an essential regulatory subunit of the Cdc7p kinase required for the initiation of DNA replication. Cdc7p and Dbf4p orthologs have also been shown to function in the response to DNA damage. A previous Dbf4p multiple sequence alignment identified a conserved approximately 40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called "motif N." BRCT motifs encode approximately 100-amino-acid domains involved in the DNA damage response. We have identified an expanded and conserved approximately 100-residue N-terminal region of Dbf4p that includes motif N but is capable of encoding a single BRCT-like domain. Dbf4p orthologs diverge from the BRCT motif at the C terminus but may encode a similar secondary structure in this region. We have therefore called this the BRCT and DBF4 similarity (BRDF) motif. The principal role of this Dbf4p motif was in the response to replication fork (RF) arrest; however, it was not required for cell cycle progression, activation of Cdc7p kinase activity, or interaction with the origin recognition complex (ORC) postulated to recruit Cdc7p-Dbf4p to origins. Rad53p likely directly phosphorylated Dbf4p in response to RF arrest and Dbf4p was required for Rad53p abundance. Rad53p and Dbf4p therefore cooperated to coordinate a robust cellular response to RF arrest.

Reduced selenium-binding protein 1 expression is associated with poor outcome in lung adenocarcinomas.

The effects of selenium, an essential nutrient with anti-carcinogenic properties, are mediated by selenium-binding proteins. The protein expression status of human selenium-binding protein 1 (SBP1) in human tumours and the exact function of this protein are not known. In this study, quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used on 93 lung adenocarcinomas and ten uninvolved lung samples. Two likely isoforms of a 56 kD protein that showed a significantly decreased abundance in lung adenocarcinomas were observed. Tandem mass spectrometry and 2-D western blot analysis identified these two proteins as human SBP1. Tumour tissue microarrays were utilized to examine the cellular expression patterns of SBP1 using immunohistochemistry. The same tissue samples were examined for SBP1 mRNA expression using oligonucleotide microarrays. Two major SBP1 isoforms were detected, with an acidic isoform (457) being significantly down-regulated in lung adenocarcinomas compared with normal lung (p = 0.02). Two additional more acidic SBP1 isoforms were only observed in normal lung. SBP1 protein isoforms and SBP1 mRNA levels were significantly decreased in poorly differentiated (versus moderately and well-differentiated), T2-T4 (versus T1), and bronchus-derived (versus bronchioloalveolar) tumours. Low levels of SBP1 protein (native form, 460) correlated significantly with poor survival (p = 0.007). The lack of SBP1 expression was not due to gene deletion. Treatment of A549 lung adenocarcinoma cells with the methylation inhibitor 5-azacytidine did not affect expression of the SBP1 protein. Analysis of the tumour proliferation status using Ki-67 suggests that down-regulated expression of SBP1 may reflect increased cell proliferation and decreased differentiation in lung adenocarcinomas.

Gene amplification in esophageal adenocarcinomas and Barrett's with high-grade dysplasia.

PURPOSE: The purpose of this study was to determine the frequency and overall contribution of specific gene amplification events in the formation of Barrett's adenocarcinomas. The relationship of gene amplification to clinical-pathological variables and its potential usefulness as a marker for early cancer detection were also examined. EXPERIMENTAL DESIGN: We used quantitative PCR and Southern blot analysis to screen 87 cases of Barrett's adenocarcinoma for the presence or absence of 13 distinct gene amplification events. Gene amplification was then examined for correlation with other amplification events and clinical variables (survival, stage, nodal involvement, tumor invasion, smoking history, and gender). Additionally, 22 specimens of Barrett's with high-grade dysplasia (HGD) were examined for the presence of gene amplification. RESULTS: One or more amplification events were present in 50 of 87 (57%) adenocarcinomas. The ERBB2 gene was amplified in 19 of 87 (21.8%), CCNE1 in 11 of 87 (12.6%), GATA4 in 9 of 87 (10.3%), KRAS in 9 of 87 (10.3%), EGFR in 7 of 87 (8.0%), CCND1 in 6 of 87 (6.8%), HNF3alpha in 5 of 87 (5.7%), PIK3CA in 5 of 87 (5.7%), C-MYC in 4 of 87 (4.6%), DYRK2 in 2 of 87 (2.3%), and AIB1, AKT1, and IGF1R were amplified in 0 of 87 (0%) of the tumors. CCND1 amplification was found to correlate negatively with survival (P < 0.05). In addition, the ERBB2 amplicon positively correlated (P < 0.05) with GATA4 amplification. Increased copy number of the ERBB2 (1 of 22), GATA4 (1 of 22), KRAS (2 of 22), C-MYC (1 of 22), CCNE1 (2 of 22), and CCND1 (2 of 22) genes was also observed in one or more Barrett's adenocarcinomas with HGD. CONCLUSIONS: The high frequency of gene amplification in esophageal adenocarcinomas and HGD indicates the important role of these events in esophageal adenocarcinoma development. Additionally, these results underscore the possible usefulness of early detection approaches and chemotherapeutic strategies (ErbB2 and cyclin D1) targeted against amplified gene products.

Increased C-CRK proto-oncogene expression is associated with an aggressive phenotype in lung adenocarcinomas.

The C-CRK gene, cellular homolog of the avian v-crk oncogene, encodes two alternatively spliced adaptor signaling proteins, CRKI (28 kDa) and CRKII (40 kDa). Both CRKI and CRKII have been shown to activate kinase signaling and anchorage-independent growth in vitro and CRKI transformed cells readily form tumors in nude mice. Affymetrix oligonucleotide arrays were used to analyse 86 lung adenocarcinomas and 10 uninvolved lung tissues. C-CRK mRNA expression was increased in more advanced (stage III versus stage I), larger (T(2-4) versus T(1)), and poorly differentiated tumors and in tumors from patients demonstrating poor survival (P=0.00034). An overlapping series of 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung specimens were measured for quantitative differences in CRKI and CRKII protein levels using 2-D PAGE. CRK protein spots were identified using mass spectrometry and 2-D Western blotting. A significant increase in levels of the CRKI oncoprotein and the phosphorylated isoform of CRKII was observed in tumors (P<0.05). No difference in protein level was evident between stages. Concordant with mRNA expression, CRKI and CRKII were increased in poorly differentiated tumors (P<0.05). CRK immunohistochemical analysis of tumor tissue arrays using the same tumor series also demonstrated increased abundance of nuclear and cytoplasmic CRK in more proliferative tumors (P<0.05). This study provides the first quantitative analysis of discrete CRKI and CRKII protein isoforms in human lung tumors and provides evidence that the C-CRK proto-oncogene may foment a more aggressive phenotype in lung cancers.

Amplification and overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) gene in esophageal and lung adenocarcinomas.

Genomic amplification can lead to the activation of cellular proto-oncogenes during tumorigenesis, and is observed in most, if not all, human malignancies, including adenocarcinomas of lung and esophagus. Using a two-dimensional restriction landmark genomic scanning technique, we identified five NotI/HinfI fragments with increased genomic dosage in an adenocarcinoma of the gastroesophageal junction. Four of these amplified fragments were matched within three contigs of chromosome 12 using the bioinformatics tool, Virtual Genome Scan. All three of the contigs map to the 12q13-q14 region, and the regional amplification in the tumor was verified using comparative genomic hybridization analysis. The 12q14 amplicon was characterized using sequence tagged site-amplification mapping with DNA from paired normal-tumor tissues of 75 gastroesophageal and 37 lung adenocarcinomas. The amplicon spans a region of >12 Mb between genes DGKA and BLOV1. The core-amplified domain was determined to be <0.5 Mb between marker WI-12457 and gene IFNG. However, MDM2, a well-documented oncogene of the region, is outside the core-domain. Eleven genes and expressed sequence tags within the amplicon were selected for quantitative reverse transcription-PCR, and DYRK2, a member of the dual-specificity kinase family, was overexpressed in all of the tumors showing gene amplification. Among the sequence tagged site/expressed sequence tag/gene markers tested, DYRK2 demonstrated the highest DNA copy number and the highest level of mRNA overexpression in the tumors. Moreover, DYRK2 mRNA overexpression (>2.5-fold of normal mean) was found in 18.6% of additional 86 lung adenocarcinomas in an assay using oligonucleotide microarrays. DYRK2 mRNA overexpression occurs more frequently than gene amplification in both esophageal and lung adenocarcinomas. This is the first report of amplification and overexpression of DYRK2 in any tumor type.

The hepatocyte nuclear factor 3 alpha gene, HNF3alpha (FOXA1), on chromosome band 14q13 is amplified and overexpressed in esophageal and lung adenocarcinomas.

Genomic amplification is observed in many, if not all, types of human malignancy and is one of the mechanisms for the activation of dominant-acting oncogenes in tumorigenesis. In the present study, three amplified restriction fragments were identified in an esophageal adenocarcinoma (P16) using the restriction landmark genome scanning two-dimensional gel technique. These fragments were cloned, sequenced, and mapped to chromosome band 14q13. Using the sequence tagged site-amplification mapping approach, we defined the core-amplified domain by screening 75 normal-tumor paired esophageal samples. The frequency of 14q13 amplification is 6.7% in esophageal tumors, and the amplicon spans >6 Mb in 1 tumor but is contained in a region <0.3 Mb in all of the remaining amplified tumors. Quantitative reverse transcription-PCR (RT-PCR) of 8 genes and expressed sequence tags located within the core-amplified domain revealed that the HNF3alpha (FOXA1)(4) gene, a forkhead gene family member, was overexpressed in all of the amplified esophageal tumors. HNF3alpha amplification was confirmed by Southern blot and interphase fluorescence in situ hybridization analyses, and the results of real-time RT-PCR were consistent with that of the regular quantitative RT-PCR. Increased immunohistochemical nuclear staining of the HNF3alpha protein was detected in all of the tumors containing 14q13 amplification. Affymetrix oligonucleotide microarrays of 86 lung adenocarcinomas demonstrated that expression of the HNF3alpha mRNA was elevated (> or =2.5-fold of mean expression in normal lung) in 37% (32 of 86) of the tumors analyzed. Gene amplification of HNF3alpha was detected in 2 of the 5 overexpressed lung tumors examined. This is the first report of HNF3alpha amplification, and overexpression in esophageal and lung adenocarcinomas. Amplification of HNF3alpha in esophageal and lung tumors may suggest a potential oncogenic role for this gene in tumorigenesis.