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"Miller, Charles T"
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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.
Journal Article
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 log 10 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.
Journal Article
Hot, Humid Air Decontamination of Aircraft Confirmed That High Temperature and High Humidity Are Critical for Inactivation of Infectious, Enveloped Ribonucleic Acid (RNA) Virus
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).
Enveloped RNA bacteriophage Phi6 (Φ6) was dried on wiring insulation, aircraft performance coating (APC), polypropylene, and nylon at ≥ 8 log
plaque-forming units (PFU) test coupon
. Only 2.4 log
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 log
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 log
and ≥5.9 log
inactivation of enveloped virus on 100 and 110 test indicators, respectively, with a 1-h treatment, excluding ramp-up and ramp-down times.
Enveloped RNA virus test indicators were successfully developed, lab tested for HHA decontamination, analyzed for RSM, and field-tested in aircraft demonstrations.
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.
Journal Article
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 ∼40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called “motif N.” BRCT motifs encode ∼100-amino-acid domains involved in the DNA damage response. We have identified an expanded and conserved ∼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.
Journal Article
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.
Journal Article
Ultraviolet Dosage and Decontamination Efficacy was 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 on to different materials for lab and field testing. Methods and Results: A live enveloped, ribonucleic acid 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) at > 8.0 log10 plaque-forming units (PFU) per test coupon. The coupons were then exposed to UV light during both lab and field testing. Commercial and prototype UV-emitting devices were measured for efficacy; 4 handheld devices, 3 room/surface-disinfecting machines, 5 air-disinfection devices, and 2 larger custom-made machines. UV device dosages ranged from 0.01-729 mJ cm-2. Anti-viral efficacy among the different UV devices ranged from no decontamination up to nearly achieving sterilization. Importantly, cardboard required far more dosage than SS304. Conclusions: 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, and prior to use. Competing Interest Statement The authors have declared no competing interest. Footnotes * Significant updates to the introduction, methods, results, discussion and references were made.
Paper
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.
Journal Article
