3M™ Petrifilm™ Rapid Yeast and Mold Count Plate Method for the Enumeration of Yeast and Mold on Selected Surfaces: AOAC Official Method 2014.05.

BACKGROUND: The 3M™ Petrifilm™ Rapid Yeast and Mold Count (RYM) Plate contains nutrients supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator system that facilitates yeast and mold enumeration in 48-60 h. OBJECTIVE: The objective of this study is to evaluate the 3M Petrifilm RYM Plate in a matrix extension study for the enumeration of yeast and mold on stainless steel, sealed concrete, and rubber surfaces. METHODS: The 3M Petrifilm RYM Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual, Ch. 18: Yeasts, Molds and Mycotoxins in a paired matrix study for enumeration of yeast and mold on stainless steel, sealed concrete, and rubber environmental surfaces. RESULTS: The 3M Petrifilm RYM Plate demonstrated equivalent performance to the reference method for enumeration of yeast and mold from stainless steel, sealed concrete, and rubber environmental surfaces. There were no significant statistical differences between the 3M Petrifilm RYM Plate and reference method results for the three matrixes evaluated. CONCLUSION: The 3M Petrifilm RYM Plate is an effective plating method for enumerating yeast and mold when analyzing stainless steel, sealed concrete, and rubber surfaces. HIGHLIGHTS: The 3M Petrifilm RYM Plate method allows the user to obtain accurate results within 48-60 h in the matrices evaluated for the presence of yeast and mold when incubated at 25 ± 1°C or 28 ± 1°C. Interpretation and colony counting was straightforward and the 3M Petrifilm RYM Plate method required no additional agar or Petri dishes, creating an easier workflow by cutting down on supplies, sample plating time, and most noticeably, occupying less space in the incubator.

3M™ Petrifilm™ Rapid Aerobic Count (RAC) Plate Method for the Enumeration of Aerobic Bacteria on Selected Surfaces: AOAC Official MethodSM 2015.13.

BACKGROUND: The 3M™ Petrifilm™ Rapid Aerobic Count (RAC) Plate is a sample-ready culture medium system designed to facilitate colony enumeration in 24 h (48 h for dry powders) for selected food and environmental surfaces. OBJECTIVE: The objective of this study is to evaluate the 3M Petrifilm RAC Plate in a matrix extension study for the enumeration of aerobic bacteria on stainless steel, sealed concrete, and rubber surfaces. METHOD: The 3M Petrifilm RAC Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual, Ch. 3 (January 2001): Aerobic Plate Count in a paired matrix study for enumeration of aerobic bacteria on stainless steel, sealed concrete, and rubber environmental surfaces. RESULTS: The 3M Petrifilm RAC Plate showed no statistically significant differences when compared to the reference method for enumeration of aerobic bacteria from stainless steel, sealed concrete, and rubber environmental surfaces. There were no significant statistical differences between the 3M Petrifilm RAC Plate and reference method results for the three matrixes evaluated. CONCLUSIONS: The 3M Petrifilm RAC Plate is an effective plating method for aerobic plate count when analyzing stainless steel, sealed concrete, and rubber surfaces. HIGHLIGHTS: The 3M Petrifilm RAC Plate is robust, quick, and simple to perform, providing enumerable colonies in 22 to 26 h after incubation when compared to the 48 h of the reference method. The small size of the Petrifilm Plate allows for less space to be occupied by plates in the incubators. The visual biochemical and enzymatic indicators make enumeration of colonies a simple task by presenting colonies in either a blue or red color.

Validation of the 3M™ Petrifilm™ Coliform Count Plate for Enumeration of Coliforms in Bottled Water: AOAC Performance Tested MethodSM 082101.

BACKGROUND: The 3M™ Petrifilm™ Coliform Count (CC) Plate is a sample-ready-culture medium system which contains modified Violet Red Bile nutrients, a cold-water-soluble gelling agent, and a tetrazolium indicator that facilitates colony enumeration. OBJECTIVE: To validate the 3M Petrifilm CC Plate method for bottled water through the AOAC®  Performance Tested Method(s)SM program. METHODS: The performance of the 3M Petrifilm CC Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4, Section III.D for the enumeration of coliforms in bottled water. Matrix data were normalized by log10 transformation and performance indicators included repeatability and difference of means with 90 and 95% confidence intervals. Inclusivity, exclusivity, robustness, and product consistency and stability were evaluated. RESULTS: This study demonstrated that the 3M Petrifilm CC Plate method detects and enumerates coliforms from bottled water. The average log10 counts of the 3M Petrifilm CC Plate method were equivalent to or better than the average log10 counts of the reference method. Results from inclusivity and exclusivity studies demonstrated that the 3M Petrifilm CC Plate method differentiated coliforms from non-coliform strains. In product consistency, stability, and robustness testing, different lots of 3M Petrifilm CC Plates and small deviations in incubation time and temperature did not affect test results. CONCLUSION: The 3M Petrifilm CC Plate method is an accurate and robust method for the enumeration of coliforms in bottled water. HIGHLIGHTS: The 3M Petrifilm CC Plate allows for detection of confirmed coliforms within 24 h. Up to 20 sample-ready plates can be stacked during incubation, providing flexibility and saving space.

Structure of the herpes simplex virus 1 genome: manipulation of nicks and gaps can abrogate infectivity and alter the cellular DNA damage response.

UNLABELLED: The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA-modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or adding random sequences to the 3' ends of DNA, producing 3' flaps, did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5' flaps in the DNA following treatment resulted in a dramatic reduction (95 to 100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5' flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5'-flapped DNA was restored in cells that do not express DNA-PKcs and in cells cotransfected with the immediate early protein ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-dependent protein kinase (DNA-PK) and the nonhomologous end joining (NHEJ) repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5' flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection. IMPORTANCE: For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest, and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes adopt a configuration suitable for lytic DNA replication. This study demonstrates that virion DNA activates the cellular DDR kinase, DNA-PK, and that this response is inhibitory to viral infection. Furthermore, we show that HSV-1 ubiquitin ligase, ICP0, plays an important role in counteracting the negative effects of DNA-PK activation. These findings support the notion that DNA-PK is antiviral and suggest that the fate of incoming viral DNA has important consequences for the progression of lytic infection. This study underscores the complex evolutionary relationships between HSV and its host.

Herpes simplex virus type 1 single strand DNA binding protein and helicase/primase complex disable cellular ATR signaling.

Herpes Simplex Virus type 1 (HSV-1) has evolved to disable the cellular DNA damage response kinase, ATR. We have previously shown that HSV-1-infected cells are unable to phosphorylate the ATR substrate Chk1, even under conditions in which replication forks are stalled. Here we report that the HSV-1 single stranded DNA binding protein (ICP8), and the helicase/primase complex (UL8/UL5/UL52) form a nuclear complex in transfected cells that is necessary and sufficient to disable ATR signaling. This complex localizes to sites of DNA damage and colocalizes with ATR/ATRIP and RPA, but under these conditions, the Rad9-Rad1-Hus1 checkpoint clamp (9-1-1) do not. ATR is generally activated by substrates that contain ssDNA adjacent to dsDNA, and previous work from our laboratory has shown that ICP8 and helicase/primase also recognize this substrate. We suggest that these four viral proteins prevent ATR activation by binding to the DNA substrate and obstructing loading of the 9-1-1 checkpoint clamp. Exclusion of 9-1-1 prevents recruitment of TopBP1, the ATR kinase activator, and thus effectively disables ATR signaling. These data provide the first example of viral DNA replication proteins obscuring access to a DNA substrate that would normally trigger a DNA damage response and checkpoint signaling. This unusual mechanism used by HSV suggests that it may be possible to inhibit ATR signaling by preventing recruitment of the 9-1-1 clamp and TopBP1.

APOBEC3 inhibits DEAD-END function to regulate microRNA activity.

The RNA binding protein DEAD-END (DND1) is one of the few proteins known to regulate microRNA (miRNA) activity at the level of miRNA-mRNA interaction. DND1 blocks miRNA interaction with the 3'-untranslated region (3'-UTR) of specific mRNAs and restores protein expression. Previously, we showed that the DNA cytosine deaminase, APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide like 3), interacts with DND1. APOBEC3 has been primarily studied for its role in restricting and inactivating retroviruses and retroelements. In this report, we examine the significance of DND1-APOBEC3 interaction. We found that while human DND1 inhibits miRNA-mediated inhibition of P27, human APOBEC3G is able to counteract this repression and restore miRNA activity. APOBEC3G, by itself, does not affect the 3'-UTR of P27. We found that APOBEC3G also blocks DND1 function to restore miR-372 and miR-206 inhibition through the 3'-UTRs of LATS2 and CX43, respectively. In corollary experiments, we tested whether DND1 affects the viral restriction function or mutator activity of APOBEC3. We found that DND1 does not affect APOBEC3 inhibition of infectivity of exogenous retrovirus HIV (ΔVif) or retrotransposition of MusD. In addition, examination of Ter/Ter;Apobec3-/- mice, lead us to conclude that DND1 does not regulate the mutator activity of APOBEC3 in germ cells. In summary, our results show that APOBEC3 is able to modulate DND1 function to regulate miRNA mediated translational regulation in cells but DND1 does not affect known APOBEC3 function.

Efficient herpes simplex virus 1 replication requires cellular ATR pathway proteins.

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.

The HSV-1 exonuclease, UL12, stimulates recombination by a single strand annealing mechanism.

Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5'-to-3' exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.

The DNA deaminase activity of human APOBEC3G is required for Ty1, MusD, and human immunodeficiency virus type 1 restriction.

Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, human immunodeficiency virus type 1 (HIV-1), were used to evaluate the relative contributions of deaminase-dependent and -independent mechanisms. Although human APOBEC3G can restrict the replication of all three of these retroelements, APOBEC3G lacking the catalytic glutamate (E259Q) was clearly defective. This phenotype was particularly clear in experiments with low levels of APOBEC3G expression. In contrast, purposeful overexpression of APOBEC3G-E259Q was able to cause modest to severe reductions in the replication of Ty1, MusD, and HIV-1(DeltaVif). The importance of these observations was highlighted by data showing that CEM-SS T-cell lines expressing near-physiologic levels of APOBEC3G-E259Q failed to inhibit the replication of HIV-1(DeltaVif), whereas similar levels of wild-type APOBEC3G fully suppressed virus infectivity. Despite the requirement for DNA deamination, uracil DNA glycosylase did not modulate APOBEC3G-dependent restriction of Ty1 or HIV-1(DeltaVif), further supporting prior studies indicating that the major uracil excision repair system of cells is not involved. In conclusion, the absolute requirement for the catalytic glutamate of APOBEC3G in Ty1, MusD, and HIV-1 restriction strongly indicates that DNA cytosine deamination is an essential part of the mechanism.

APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast.

Human cells harbor a variety of factors that function to block the proliferation of foreign nucleic acid. The APOBEC3G enzyme inhibits the replication of retroviruses by deaminating nascent retroviral cDNA cytosines to uracils, lesions that can result in lethal levels of hypermutation. Here, we demonstrate that APOBEC3G is capable of deaminating genomic cytosines in Saccharomyces cerevisiae. APOBEC3G expression caused a 20-fold increase in frequency of mutation to canavanine-resistance, which was further elevated in a uracil DNA glycosylase-deficient background. All APOBEC3G-induced base substitution mutations mapped to the nuclear CAN1 gene and were exclusively C/G --> T/A transition mutations within a 5'-CC consensus. The APOBEC3G preferred sites were found on both strands of the DNA duplex, but were otherwise located in hotspots nearly identical to those found previously in retroviral cDNA. This unique genetic system further enabled us to show that expression of APOBEC3G or its homolog APOBEC3F was able to inhibit the mobility of the retrotransposon Ty1 by a mechanism that involves the deamination of cDNA cytosines. Thus, these data expand the range of likely APOBEC3 targets to include nuclear DNA and endogenous retroelements, which have pathological and physiological implications, respectively. We postulate that the APOBEC3-dependent innate cellular defense constitutes a tightly regulated arm of a conserved mobile nucleic acid restriction mechanism that is poised to limit internal as well as external assaults.

APOBEC3F properties and hypermutation preferences indicate activity against HIV-1 in vivo.

APOBEC3G (CEM15 ) deaminates cytosine to uracil in nascent retroviral cDNA. The potency of this cellular defense is evidenced by a dramatic reduction in viral infectivity and the occurrence of high frequencies of retroviral genomic-strand G --> A transition mutations. The overwhelming dinucleotide hypermutation preference of APOBEC3G acting upon a variety of model retroviral substrates is 5'-GG --> -AG. However, a distinct 5'-GA --> -AA bias, which is difficult to attribute to APOBEC3G alone, prevails in HIV-1 sequences derived from infected individuals (e.g., ). Here, we show that APOBEC3F is also a potent retroviral restrictor but that its activity, unlike that of APOBEC3G, is partially resistant to HIV-1 Vif and results in a clear 5'-GA --> -AA retroviral hypermutation preference. This bias is also apparent in a bacterial mutation assay, suggesting that it is an intrinsic APOBEC3F property. Moreover, APOBEC3F and APOBEC3G appear to be coordinately expressed in a wide range of human tissues and are independently able to inhibit retroviral infection. Thus, APOBEC3F and APOBEC3G are likely to function alongside one another in the provision of an innate immune defense, with APOBEC3F functioning as the major contributor to HIV-1 hypermutation in vivo.