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Following a referendum on 23 June 2016, 51.9% of UK voters said “yes” to Brexit. British Prime Minister Theresa May revealed earlier this month that Article 50 TEU will be triggered by March 2017, marking the beginning of the formal withdrawal process.
It is hard to predict the impact of Brexit on the current copyright framework, since much will depend on the agreement reached between the EU and the UK. Several options are possible, from a custom-made agreement to an EFTA or EEA type of agreement; or even no agreement at all, which would make the UK a “third country” vis-à-vis the EU. Each of these solutions entails different consequences for both parties involved.
From a political perspective, it seems that a clash is on the horizon: the UK is likely to want to negotiate a “cherry pick agreement”, i.e., an agreement where it gets to pick and choose the EU rules it wants; whereas the EU will probably prefer an “all-or-nothing” approach. Moreover, in the field of intellectual property, and if the free trade agreements negotiated by the EU with third countries are anything to go by, it can be expected that the EU will want to export its intellectual property standards, enforcement included. How and to what extent the EU will be willing to deviate from this practice is unknown. But to this political context it should be added that, according to Article 50 para. 2 TEU, the agreement with the UK is to follow certain guidelines provided by the EU Council, which in turn is composed of the heads of state or government of the Member States. The political indigestion created by Brexit across Europe might not favour the UK when the time comes to negotiate the terms of the Brexit agreement. It is thus difficult to foretell to what extent the UK will manage to get a custom-made agreement – in copyright and beyond. Part of the consideration below is therefore dependent on that missing part of the puzzle.
Should the UK not be obliged to maintain its laws in line with the EU copyright acquis, it might take the opportunity to “customise” its copyright law. The precise way in which the UK will be able to do so is however unclear. The EU copyright directives have been implemented by Statutory Instruments– a legislative instrument of secondary nature that depends on a “parent Act”, in this case the European Communities Act of 1972 (by which the UK joined the European Community). The British Secretary of State for Exiting the European Union has announced plans for a “Great Repeal Bill”, which would repeal the European Communities Act. This would repeal the legislation enacted under it (read: all the statutory instruments implementing the EU Directives), unless the “Great Repeal Bill” expressly saves subordinate legislation (as explained excellently and at length here). What is saved, if anything, is yet another unknown element, and most likely highly dependent on political compromises.
With those notes in mind, we can think of a few examples that illustrate the difficult decisions ahead. Let’s take, for instance, the sui generis right for databases. Would this be a serious contender for the title of EU relic within a new UK copyright law? On the one hand, databases (both content- and structure-wise) were already protected to a considerable extent in the UK prior to the Database Directive, due to the (lower) British threshold for protection. In addition, the European Commission acknowledged that the economic effect of the sui generis right is unproven, and earlier this year the European Parliament called on the Commission to abolish the Database Directive. Given Europe’s own scepticism regarding the Database Directive, and since the UK has a pre-Directive tradition of database protection to fall back on, a return to a pre-Directive scenario would thus not be as far-fetched as it might seem at first sight. On the other hand, the return to an untouched “skill, labour or judgment” threshold will depend as well on how UK Courts apply the originality criterion in a post-Brexit world (which is both a challenge, and a customisation opportunity, in and of itself). Moreover, and perhaps more importantly, the UK is the most productive database maker in relative terms (as shown in the First Evaluation of the Database Directive). That being the case, the UK might not want to risk their databases being ineligible for sui generis protection in the EU (which could happen under the reciprocity clause of the Directive if the UK gets a “third country” type of deal, again of course dependent on the terms of a potential agreement in that regard). Access to the EU market and uncertainty regarding the threshold of protection absent the sui generis right might therefore mean that the UK will want to keep the very European sui generis protection for its databases.
Another example is the resale right. The UK was a strong opponent to the introduction of a resale right, one of the reasons being fear of displacement of art sales to third countries that didn’t have a resale right. Even though no evidence of displacement of art sales was found, the case remains that British stakeholders are divided in their opinion of the resale right, with some (e.g., art dealers) claiming that it has negative effects, while others (such as artists) have a positive outlook on it. With whom a potential new UK copyright law would side is a complex issue to predict.
Many more examples of prospects for the UK to customise its copyright law could be found. Judging from the ones mentioned above, however, it can be argued that the opportunity to customise UK copyright law also poses considerable challenges. The introduction of EU rules in the UK legal order has brought about benefits to (at least) some factions of British society. Justifying getting rid of those “European perks” to their respective beneficiaries might be a tough task. Not getting rid of them, on the other hand, might meet opposition by stakeholders not deriving any advantage from EU rules, and from the crowd of Brexiteers. Decisions on how to customise copyright law, if they are at all possible, might thus come down to a question of political convenience and lobbying strategies – much to the detriment of the copyright system as a whole, as well as its users.
Yet another interesting point of discussion is the relevance of the case law of the CJEU. What will be the impact of both pre-Brexit and post-Brexit CJEU case law on the UK legal order? In relation to the former, some past examples illustrate a certain British resistance to accepting CJEU jurisprudence tout court. For instance, with regard to the definition of originality, it has taken UK courts a few decisions to internalise the EU threshold of “author’s own intellectual creation” as advanced by the CJEU in Infopaq (see the hesitation of the British court in using the CJEU threshold, e.g., in Meltwater or in Taylor v Maguire).
Whether UK courts will take the opportunity to go back to the previous British definition of originality (and other pre-CJEU British interpretations of EU directives) remains to be seen – not least because it is still unclear which parts of EU copyright will be kept in UK copyright law. The reason to follow previous CJEU case law (EU membership) ceases to exist, but it is possible that, for reasons of legal certainty, the UK courts will choose to stick to the CJEU interpretations already incorporated in their rulings. It is also possible that the “Great Repeal Bill” or ensuing laws that save particular aspects of EU copyright law establish rules on precedent and interpretation of those EU law rules that the UK decides to keep.
CJEU case law post-Brexit will in principle cease to be binding on the UK, unless e.g. a future agreement between the UK and the EU includes compulsory jurisdiction of the CJEU over the remaining aspects of EU law in the UK legal system. Regarding such remaining aspects, and absent a binding force of CJEU case law, it will be interesting to observe how the same (EU) copyright rules will evolve on the two sides of the Channel as a result of different judicial interpretations (i.e., by the CJEU, on the one hand, and by British Courts, on the other).
Brexit will also mean that the UK will now be entirely responsible for concluding international agreements and treaties that cover the commercial aspects of intellectual property, including copyright. A few previous agreements concerning intellectual property, such as TRIPS, have been entered into by both the EU and the UK. Other instruments, however – notably, free trade agreements with non-EU countries, many of which include intellectual property provisions – have been entirely concluded by the EU. After Brexit, the UK will no longer be party to those and will have to negotiate new agreements with said countries – presumably with much less bargaining power.
The first foreseeable consequence of the UK leaving is that, without the main representative of common law systems, the EU might inflect towards a civil law approach to copyright law. With the UK gone, the need for political compromise between civil and common law solutions will no longer have the same weight. For example, and coming back to the question of originality, its definition as the “author’s own intellectual creation” in the Computer Programs Directive has, as the Commission admitted, “required 12 Member States to lower the threshold for granting protection and the remaining three to “lift the bar””. This denotes the compromise character of the originality criterion, to which surely the weight of the UK on the side of the lower threshold for protection contributed. In other words, the UK’s influence in achieving common ground solutions between different legal traditions is not to be disregarded; in the battle between civil law and common law influences, the odds are now tilted towards a victory of the former.
Still, this probably won’t result in a harmonisation of too-hot-to-handle potatoes, such as moral rights (also because their connection to the building of an internal market can be disputed). The same goes for copyright contracts. Even though the UK is a strong advocate for freedom of contract (and therefore adverse, in principle, to regulating this area of copyright law), an EU without the UK would not necessarily proceed to harmonising Member States’ laws on copyright contracts: the marked differences between the remaining 27 national laws would require a level of political agreement that just does not seem plausible.
A related question is whether, with the UK gone, the EU might be one step closer to having a unified copyright title. In its contribution to the public consultation on the review of EU copyright rules (2014), the UK advised the Commission against “rushing into” an EU single copyright title. A similar stance had been taken back in 2011, regarding the consultation on the Green Paper on the online distribution of audio-visual works, where the UK showed a preference for looking into other legislative and non-legislative options before considering an EU copyright code. However, to be fair, other Member States, such as Slovakia, Italy, Poland, or Latvia, argued against the creation of an EU single copyright title in their respective contributions to the 2014 consultation – which might indicate that the “no-code” team lost a player, but not the game.
Another point to consider is the EU rule on exhaustion of the distribution right. This would not be a problem should the UK get an EEA status, as the exhaustion rule applies there. The same goes for the case where an exhaustion rule is negotiated between the UK and the EU in a custom-made agreement (or in an agreement whereby the UK vows to keep the laws implementing the directives containing the exhaustion rule). However, if none of these scenarios occur (e.g., if the UK gets a third-country status with no agreement on this point), this would mean that copyright protected goods first placed on the UK market would not exhaust the distribution right of the right holders (see on this Recital 28 of the InfoSoc Directive and Laserdisken). The right holder could therefore oppose the import of such goods into the EU (much to the detriment of trade of copyright goods in and out of the UK).
Many other copyright issues deriving from Brexit could be outlined, from either point of view. The EU will, without a doubt, miss its main common law player. But, all in all, it seems that in this complicated divorce the UK will face the bigger challenge. Many aspects of EU copyright law are imprinted on the UK’s own copyright system, and untangling them will require considerable amounts of skill and labour (no pun intended).

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Flying an unmanned aerial system (UAS) – also known as a drone – is not illegal in the state of Florida. It is very possible though to commit a crime that is not aimed at drones while flying one.
These crimes consist of misdemeanors, and even felonies. People sometimes think that they can just avoid a criminal record by pleading “no contest” or accepting probation. If you were charged or cited with a misdemeanor, you should not attempt to resolve it without the assistance of an experienced criminal defense attorney. A felony conviction can have devastating consequences on your life and can reduce chances of finding gainful employment or furthering your education and the assistance of an attorney is even more important.
If you don’t do things right, you might get convicted of the crime and face very serious collateral consequences. It is important to consult with an attorney – not only who has a proven track record in criminal defense – but who also understands the rights of drone pilots under Florida law and FAA regulations.
If you or someone you know has been charged with an infraction using a drone, it is imperative to hire an experienced attorney who can help reduce your charges or seek a minimal sentence. To potentially avoid very harsh punishments, contact the attorneys at the Pumphrey Law at (850) 681-7777 for a free consultation regarding your alleged offense.
The Pumphrey Law has a great deal of experience representing Florida residents accused of many different types of misdemeanor and felony offenses.
Drone laws are still in their formative stages in Florida, but nationwide people have already been arrested for crimes while flying a drone, or in reaction to someone else flying one. The FAA estimated that there were 2.5 million drones in the sky, set to almost double by the conclusion of 2017. These estimates are set to skyrocket to 7 million by the end of the decade. As more and more UASs take to the skies, more and more people are going to be arrested for pushing the boundaries.
If you’re going to fly a drone, you always need to make sure that you’re following FAA rules, whether you’re flying for fun, or if you’re using it to make money. This doesn’t necessarily mean that you’re going to be ok under Florida Law though.
3. Allow a drone to come within a distance of a critical infrastructure facility that is close enough to interfere with the operations of or cause a disturbance to the facility.
Drug Trafficking – Moving drugs via drone is illegal, and a conviction is at least a first-degree felony according to Florida Statutes § 893.135.
Stalking or Aggravated Stalking – Using a drone to stalk someone is exactly the same as stalking without a drone, and could be anywhere from a misdemeanor in the first degree to a felony in the third degree according to Florida Statutes § 784.048.
Assault – Threatening to hit someone with a drone could be considered assault, a misdemeanor in the second degree according to Florida Statutes § 784.011. If the court chooses to allow a drone to be considered a “deadly weapon,” this could even rise to aggravated assault, a felony in the third degree according to Florida Statutes § 784.021.
Battery – Hitting someone with a drone would likely be battery, a misdemeanor in the first degree, or felony in the third degree after the first offense, according to Florida Statutes § 784.03.
Destruction of Aircraft/Endangering the Safety of Pilot – Drone users aren’t the only ones who can get arrested while interacting with a drone. Damaging or destroying a UAS is a federal crime according the FAA because UASs are considered aircraft. Further, it is a crime to attack “anyone engaged in the authorized operation of such aircraft.” The FAA believes this applies to UAS pilots. Breaking either of these laws is a federal crime, and a conviction means a fine, up to 20 years in federal prison, or both, according to 18 U.S.C. § 32.
Aside from drone crimes, there are also various civil infractions that drone pilots face.
If you have been charged with a crime while using a drone in Florida, contact the attorneys at the Pumphrey Law to discuss the facts of your particular situation. There may be defenses to your charge, and finding an attorney who will aggressively fight on your behalf is your best option to avoid severe punishment.
The attorneys at the Pumphrey Law are experienced in criminal defense, and may be able to reduce or alleviate your charges completely. Contact us at (850) 681-7777 for a consultation about your alleged offense.
This article was last updated November 11, 2017.
 Fla. Stat. § 330.41 (2)(a) (“‘Critical infrastructure facility’ means any of the following, if completely enclosed by a fence or other physical barrier that is obviously designed to exclude intruders, or if clearly marked with a sign or signs which indicate that entry is forbidden and which are posted on the property in a manner reasonably likely to come to the attention of intruders”).


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We report the sequence-based characterization and expression patterns of three manganese peroxidase genes from the white rot fungus and grape vine pathogen Fomitiporia mediterranea (Agaricomycotina, Hymenochaetales), termed Fmmnp1, Fmmnp2, and Fmmnp3. The predicted open reading frames (ORFs) are 1,516-, 1,351-, and 1,345-bp long and are interrupted by seven, four, and four introns, respectively. The deduced amino acid sequences encode manganese peroxidases (EC 1.11.1.13) containing 371, 369, and 371 residues, respectively, and are similar to the manganese peroxidases of the model white rot organism Phanerochaete chrysosporium. The expression of the genes is most likely differentially regulated, as revealed by real-time PCR analysis. Phylogenetic analysis reveals that other members of the order Hymenochaetales harbor mnp genes encoding proteins that are related only distantly to those of F. mediterranea. Furthermore, multiple partial lip- and mnp-like sequences obtained for Pycnoporus cinnabarinus (Agaricomycotina, Polyporales) suggest that lignin degradation by white rot taxa relies heavily on ligninolytic peroxidases and is not efficiently achieved by laccases only.
Fungal class II peroxidases have been studied extensively since their discovery and initial characterization in the lignin-degrading model organism Phanerochaete chrysosporium (20, 65). They belong to the larger superfamily of non-animal peroxidases (47), originally described and termed the plant peroxidase superfamily by Welinder (67). The three-dimensional structure is strongly conserved among all known class II enzymes (2). Functional aspects of these enzymes have been studied in detail in P. chrysosporium (12, 37, 66). Fungal class II peroxidases can be divided into four groups based on the preferred substrates oxidized and the presence of key substrate-binding amino acid (aa) residues. Lignin peroxidases (LiPs) (EC 1.11.1.14) catalyze the oxidation of phenolic and nonphenolic aromatic compounds, such as synthetic lignins, by cleaving the Cα-Cβ ether bonds but also act on the fungal metabolite veratryl alcohol (37). The reaction mechanism involves a long-range electron transfer pathway initiating from an exposed tryptophan residue (12, 48). Essentially the same pathway is present in versatile peroxidases (VPs) (EC 1.11.1.16), but additionally these enzymes contain three Mn-binding residues (9). Oxidation of Mn2+ to Mn3+ had been previously described as the major function of manganese peroxidases (MnPs) (EC 1.11.1.13), in which the same Mn-binding residues are present (18). Chelated Mn3+ ions are thought to extract electrons from phenolic aromatic compounds directly or via lipid peroxidation from nonphenolic aromatics as well (19, 40). There is significant evidence that the enzymes LiP, VP, and MnP act as ligninolytic enzymes, although some questions regarding the mechanism of lignin degradation still remain open (22). Functionally less well characterized is a fourth group of class II peroxidases represented by the Coprinopsis cinerea peroxidase (CiP) and the hybrid or novel peroxidase of P. chrysosporium (nop or hip). These enzymes are characterized as low-redox peroxidases and are considered to be nonligninolytic (54).
Due to the potential biotechnological applications of ligninolytic peroxidases with respect to the improvement of feedstock quality for the production of second-generation biofuels, there has been an increased interest in the study of these secreted proteins. White rot fungi, which have the unique ability to degrade lignin, secrete these enzymes under ligninolytic conditions, which include nitrogen and carbon limitation (34, 60). Under laboratory conditions, heat shock, hydrogen peroxide concentration, oxygen supplementation, and growth medium characteristics (solid versus liquid, agitated versus stationary) influence the level of enzyme production (8, 17, 33, 53). MnP production depends on sufficient levels of Mn2+ in the medium (7).
Much progress has been achieved in recent years regarding the evolution of the fungal peroxidase family, partly due to genome projects but also due to continued research interest in this family in non-model organisms. Phylogenetic analyses revealed that VPs arose independently at least twice, that LiPs are monophyletic and are presently known only from the Polyporales, and that MnPs are the most widespread ligninolytic peroxidases (41, 55). From an evolutionary perspective, two types of MnP can be distinguished. One type gave rise to LiPs and VPs, sharing a high degree of overall similarity with them, as is evident in protein length (354 to 365 residues; average, 363), exon-intron pattern (5 to 16 introns; median = 11), and the number of disulfide bonds (four cystines). The second type of MnP is longer (376 to 395 aa; average = 383), has fewer introns (6 to 13; median = 6), and is characterized by an additional disulfide bond, presumably to stabilize the longer C terminus (62). Members of these “classical” or long MnPs form a monophyletic group that branches off early from the phylogenetic tree and contains a putative MnP sequence from Fomitiporia mediterranea, a member of the Hymenochaetales, in addition to sequences from the Polyporales and Corticiales (41). So far, sequences for MnPs, LiPs, and VPs have been detected only in wood-degrading white rot taxa, with the exception of an MnP from Agaricus bisporus, which is a leaf litter- and compost-degrading saprotroph. Class II peroxidases of yet unknown function are present in some brown rot taxa (25, 36) and in a diversity of ectomycorrhizal (ECM) taxa (4, 35).
In contrast to the Polyporales and Agaricales, other fungal taxonomic groups containing white rot taxa have been less well studied with respect to their ligninolytic peroxidase systems. The Hymenochaetales are a major order of the Agaricomycotina situated in a more basal position than the Polyporales and Agaricales (23). Members of this clade are almost exclusively wood-decaying white rot fungi and confirmed brown rot fungi are not present, but a few cases of ectomycorrhizal symbionts have been reported (31). Decay activity is usually described as mostly saprotrophic, but some species, notably Porodaedalea pini and Phellinus igniarius, qualify as timber pathogens (31). Two Phellinus species demonstrably decolorize textile dyes, a trait commonly attributed to phenoloxidase activity, but the actual enzymes involved have not been identified (1, 45).
F. mediterranea is a member of the Hymenochaetales that has an Old World distribution, where it occurs mainly on Vitis vinifera (16) and a few other host plants (14, 15, 63). The association between F. mediterranea and V. vinifera is of particular relevance, since the fungus produces an extensive white rot in the trunks of growing Vitis plants, which is one symptom of the destructive grapevine disease complex esca, affecting vine cultivation on a global scale (51). It is not yet clear if F. mediterranea acts independently from other pathogens or if the observed white rot decay is the result of favorable conditions created by a primary infection with ascomycetes (42). Surprisingly, expression of MnP and LiP in connection with the esca disease has been attributed to Phaeoacremonium aleophilum (anamorph of Togninia minima) (11, 43), and an MnP-like peroxidase was reported for Phaeoacremonium viticola and Phaeoacremonium angustius (46).
The purpose of our study was to determine if species in the Hymenochaetales have a reservoir of ligninolytic class II peroxidases that is comparable to enzymes characterized in the Polyporales or Agaricales. To achieve this we obtained additional partial peroxidase sequences for F. mediterranea and new sequences for other members of the Hymenochaetales. A further goal was to obtain the full-length sequences for F. mediterranea class II peroxidase genes and characterize them in a comparative way with the sequences from other lignin-degrading taxa. Finally, we monitored mRNA transcript abundance for mnp genes from F. mediterranea grown in liquid culture supplemented with different manganese concentrations.
Strain and culture conditions.A single spore isolate from F. mediterranea strain MF 3/22 was maintained on malt extract (ME) medium (2% malt extract, 2% agar, 0.05% yeast extract) in petri dishes and regularly subcultivated. Prior to DNA extractions, ca. 0.5-cm by 0.5-cm mycelium plaques from the mycelial growth zone on the surfaces of the agar plates were removed aseptically and transferred into 500-ml Erlenmeyer flasks containing 100 ml liquid ME medium (2% malt extract, 0.05% yeast extract). For RNA extractions, cultures were treated similarly but inocula were transferred into six-well Falcon plates (Becton Dickinson Labware, Franklin Lakes, NJ) containing 7.5 ml liquid ME medium per well. Cultures were grown without agitation under ambient conditions. When the cultures in the well plates covered approximately 80% of the surface area, the full nutrient medium was removed and the cultures were washed by being rinsed three times with distilled water, and after removal of excess liquid, 5 ml of the assigned experimental minimum salts medium was added to the cultures. Basal minimum salts medium contained the following per liter of distilled water: 0.2 g KH2PO4, 0.05 g MgSO4·7 H2O, 0.01 g CaCl2, 10 g glucose (56 mM) as a carbon source; 2.9 g ammonium tartrate (1.2 mM N) as a nitrogen source; 2.92 g sodium-2,2-dimethylsuccinate buffer (20 mM), pH 4.5; 1 ml mineral solution; and 1 ml vitamin solution (ICN Pharmaceuticals) (30). Mineral solutions were essentially the same as those described in reference 30 but were prepared separately for each experimental condition (zero/low/high Mn levels), differing only in the amount of MnSO4·H2O added. The final Mn concentration in the growth medium was 60 μM for the high-Mn concentration treatment, 6 μM for the low-Mn concentration treatment, and 0 μM for manganese-free treatment.
DNA and RNA extractions.Genomic DNA from F. mediterranea cultures was extracted from 3 to 5 g fresh material after vacuum filtration using a maxi-prep protocol (http://www.clarku.edu/faculty/dhibbett/protocols.html) and purified with the Geneclean II kit (Bio 101, La Jolla, CA), quantified spectrophotometrically, and stored at −20°C. On days 1, 3, 5, and 7 after medium exchange, RNA was extracted from 90 mg fresh material after vacuum filtration using the Qiagen RNeasy mini kit (Valencia, CA) and mechanical disruption using a Mini-Beadbeater-8 (BioSpec Products, Bartlesville, OK). Mycelia were homogenized by twice disrupting the cells for 4 min each and interrupted by cooling the material on ice for a few minutes. Purified RNA was stored at −80°C.
Standard PCR and sequencing analyses.Newly generated partial sequences for this study were obtained as previously described using primer combinations aVP-1F/bLP-7R and hymMP-f/hymMP-r (41). We focused on members of the Hymenochaetales but included also members of the Polyporales. Thermal cycle parameters were as follows: a 2-min initial denaturation at 95°C, 30 cycles of 30-s denaturation at 94°C, 30 s of annealing at 55°C, a 1-min extension at 72°C, and a 10-min final extension at 72°C. Purification and molecular cloning of amplification products was done as described in a previous study (41). Sequencing was carried out on an ABI Prism 3130 genetic analyzer (Applied Biosystems, Foster City, CA).
IPCR analysis.Genomic DNA was digested with the restriction enzymes MseI, NspI, ApoI, and HindIII (New England Biolabs). Restriction digests were carried out with 2.0 to 3.0 μg total genomic DNA for 3 h at optimum temperatures (37°C for MseI, NspI, and HindIII and 50°C for ApoI) in 50-μl reaction volumes and 20 U of restriction enzyme per reaction mixture. After purification (Qiagen), digested DNA was diluted 5-fold and ligated in a 50-μl reaction volume using 40 U of T4 DNA ligase (New England Biolabs) at 16°C for 16 h. For each sample DNA, three ligation reactions were carried out with increasing amounts of template (5 μl, 10 μl, 25 μl). First-round inverse PCR (IPCR) was carried out using 5-μl aliquots from the ligation reaction, 12.5 pM of each inner IPCR primer (Table 1), 0.2 mM each deoxynucleoside triphosphate (dNTP), 2.5 μl 10× reaction buffer, and 1.25 units Taq DNA polymerase in a 25-μl reaction volume. First-round PCR conditions were as follows: initial denaturation at 95°C for 2 min, followed by 9 cycles of denaturation at 94°C, annealing in a touchdown approach starting at 60°C and ending at 52°C (−1°C per cycle) for 40 s, and extension at 72°C for 2 min 15 s, followed by 37 cycles of denaturation at 94°C for 45 s, annealing at 52°C for 1 min 30 s, and extension at 72°C for 2 min 15 s, followed by a final extension at 72°C for 15 min. Amplification products were column cleaned (Qiagen) and subjected to second-round PCR analysis with nested outer inverse PCR primers. A 5-μl aliquot from the first-round PCR was used as a template in the second-round PCR in a total reaction volume of 25 μl. Second-round amplification parameters consisted of initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min 15 s, followed by a final extension at 72°C for 10 min. Amplification results were visualized with ethidium bromide staining of a 1% agarose gel after gel electrophoresis of an aliquot. Reaction results producing a single band were purified (Qiagen) and cloned using the TOPO TA cloning kit (Invitrogen).
RT-PCR and real-time PCR analyses.Transcript levels of the three newly characterized mnp genes in F. mediterranea were measured in comparison to the transcript levels of the housekeeping gene ef-1α and are interpreted as relative expression levels. First-strand cDNA was synthesized from 0.1 μg total RNA using the QuantiTect reverse transcription (RT) kit (Qiagen) according to the manufacturer's guidelines. Genomic DNA was eliminated from the samples by incubation of samples for 4 min at 42°C with the supplied genomic DNA (gDNA) wipeout buffer prior to reverse transcription. For control purposes, each sample was also reverse transcribed without reverse transcriptase added to the master mix. Second-strand cDNA generation was obtained by using real-time PCR analysis on a Stratagene Mx3000P thermal cycler. Real-time experiments were conducted in triplicate from each biological sample using the Brilliant II SYBR green quantitative PCR (qPCR) master mix (Stratagene) with final primer concentrations of 0.2 μM (ef-1α-specific primers) and 0.3 μM (all mnp-specific primers) per reaction. Amplification conditions were as follows: 10 min at 95°C, 40 cycles of 30 s at 95°C, and 1 min at 60°C, followed by a final dissociation cycle with 1 min at 95°C, 30 s at 55°C, and a ramp phase of up to 95°C with a 0.2°C increase per second. The mean normalized expression was calculated using the Q-Gene module (http://www.gene-quantification.de/download.html) by averaging the three threshold cycle (CT) values of each replicate and subsequent calculation of the mean normalized expression (44). Accordingly, the standard error was calculated using the differential equation of Gauss for error propagation. The amplified products were run in 1.6% agarose gels for size comparison, purified, cloned as described above, and sequenced to verify specificity of the amplicons obtained from qRT-PCR.
Phylogenetic analyses.The three full-length mnp sequences from F. mediterranea and 22 partial class II peroxidase sequences generated by this study were added to a preexisting data set (41). We amplified partial sequences for Phylloporia ribis, Inonotus hispidus, Hymenochaete corrugata (Hymenochaetales), Pycnoporus cinnabarinus, Grifola frondosa, and Steccherinum fimbriatum (Polyporales). Previously, no class II sequences were available for any of these white rot taxa. An additional 16 class II peroxidase sequences, mostly from ectomycorrhizal taxa but also including some from the white rot fungus Phellinidium ferrugineofuscum (Hymenochaetales), were obtained from a recent study (4). BLASTP searches against the NCBI GenBank detected 21 more sequences. After pruning redundant sequences, the final data set contained 177 sequences (77 full length and 100 partial). Multiple sequence alignment was performed with MAFFT using the E-INS-i setting (28, 29). The signal sequence and one ambiguous region present only in the two outgroup sequences were excluded from the analyses. Bayesian analysis was run for 10 million generations with eight Markov chain Monte Carlo (MCMC) chains in two runs, each with 1 cold and 3 heated chains for 10 million generations, mixed model for amino acid substitutions, and trees sampled every 1,000 generations using MrBayes v3.1.2 (26, 52). Convergence of likelihood scores was established with Tracer v1.4 (http://beast.bio.ed.ac.uk/Tracer), and the first 3,000 trees from each run were removed as the burn-in before calculating the consensus tree. Maximum likelihood (ML) analyses were performed using RAxML (59) with the rapid hill-climbing algorithm and the WAG substitution model. A tree with the best likelihood scores was obtained from an analysis combining the search for the best ML tree with the rapid bootstrap analysis using 100 bootstrap replicates, and final bootstrap support was estimated from a run with 1,000 bootstrap replicates.
Characterization of three F. mediterranea mnp genes.Using standard and inverse PCR analyses, we obtained the full-length sequences of three putative mnp genes from F. mediterranea. Putative intron-exon boundaries as well as start and stop codons were predicted based on homologous sequences. The leader sequence was identified using SignalP 3.0 (3). Accordingly, gene Fmmnp1 consists of a 1,516-bp open reading frame (ORF) interrupted by seven introns (see Fig. S1a in the supplemental material). The deduced amino acid sequence of the prepropeptide is 371 aa long and includes a 18-aa signal peptide, followed by a 5-aa propeptide leader sequence that ends with a KEX2 motif (Lys-Arg). The mature protein is predicted to contain 348 aa. Fmmnp2 (see Fig. S1b) has a 1,351-bp-long ORF and is interrupted by four introns. The amino acid sequence of the prepropeptide encoded by Fmmnp2 is 369 aa long, including a 17-aa-long signal peptide and 5 aa with the KEX2 motif preceding the mature peptide. The ORF of Fmmnp3 is 1,345 bp long (see Fig. S1c). The predicted coding sequence is interrupted by four introns. The prepropeptide sequence is 371 aa long and contains a 19-aa-long signal sequence and the 5-aa propeptide leader sequence. The presence of a KEX2 cleavage motif is common among most class II peroxidases, but it is not present in the long MnPs from the Polyporales (Fig. 1, clade MnP1, without Hymenochaetales). MnPs from the Hymenochaetales seem to have retained this feature before it was lost in the lineage leading to the Polyporales. However, MnPs from F. mediterranea share with the long MnPs from P. chrysosporium and other Polyporales (Fig. 1, clade MnP1) two additional cysteine residues, allowing the formation of a fifth disulfide bond. These cysteine residues are 7 aa apart and their positions are well conserved. However, in F. mediterranea only isoform MnP1 cysteines occupy exactly the same positions; in isoforms MnP2 and MnP3, the distance between the cysteine residues is 9 aa. The stop codon in the F. mediterranea MnPs (FmMnPs) follows only three and four residues after the last cysteine, whereas other MnPs in clade MnP1 have between 9 to 21 residues before the stop codon, giving the mature protein a considerably longer C-terminal tail. Residues important for peroxidase function, which are conserved throughout the peroxidase superfamily, are present in the three F. mediterranea MnPs, and include the distal and proximal histidine and other residues involved in heme pocket formation. Mn-binding residues and putative N glycosylation sites (consensus sequence N-X-T/S) have been identified and are indicated (see Fig. S1a to c in the supplemental material). The calculated isoelectric points and molecular masses for the holoenzymes are as follows: pI 4.71, 39.3 kDa for FmMnP1; pI 4.25, 38.7 kDa for FmMnP2; and pI 4.52, 39.1 kDa for FmMnP3. Fomitiporia mediterranea MnPs lack the exposed tryptophan residue characteristic for LiPs and VPs; however, this position is uniquely occupied by a histidine residue in FmMnP2 and FmMnP3 and in other partial sequences from hymenochaetalean taxa. Predicted introns ranged in size from 51 to 63 nucleotides (mean, 58), and all followed the canonical GT-AG splice rule. In contrast to the P. chrysosporium mnp genes that have a remarkably conserved exon-intron pattern, none of the F. mediterranea introns is present in all three mnp genes. A comparison with other mnp genes and basal peroxidases reveals that the intron positions in F. mediterranea are more similar to those in basal peroxidases and Lentinus edodes mnp2 than to those in mnp genes from the Polyporales (Fig. 2). We found several unique predicted introns in Fmmnp1 that are not shared with any other peroxidase used in the comparison.
Maximum likelihood tree showing phylogenetic relationships of class II fungal heme peroxidases. ML bootstrap support values above 70 are indicated before the slash. Bayesian posterior probability values above 0.9 are indicated after the slash. Fungal orders are distinguished by colored boxes. ‡, ectomycorrhizal taxa. Sequences generated by this study are in bold. Labeling of clades and clusters takes phylogenetic considerations and structural-functional aspects into account, which are based on the presence of crucial residues in the protein sequence. Accordingly, MnPs are characterized by the presence of the Mn-binding site (three acidic residues) and the absence of an exposed tryptophan. VPs are characterized by the presence of the Mn-binding site and the exposed tryptophan. LiPs are characterized by the presence of the tryptophan and the absence of the Mn-binding site. Some partial sequences do not cover all three Mn-binding residues and/or the exposed tryptophan.
Comparison of intron distribution for the three new F. mediterranea peroxidase genes with 10 selected full-length sequences from clade MnP1 and basal peroxidases. Introns are shown as black bars, signal peptides are shaded dark gray, and introns shared between F. mediterranea and other taxa are light gray. Cc, Coprinopsis cinerea; Ds, Dichomitus squalens; Fm, Fomitiporia mediterranea; Le, Lentinus edodes; Pc, Phanerochaete chrysosporium; Pp, Postia placenta; Pr, Phlebia radiata.
Comparison of relative expression levels.Quantitative RT-PCR experiments resulted in a single product for each primer pair evident from dissociation curve analyses as well as from cloning and sequencing of the obtained RT-PCR amplicons, confirming expression of the three putative mnp genes in F. mediterranea. The transcript abundance for each isoform varied, depending mostly on the time after nutrient exchange and to a lesser degree on the manganese concentration in the medium. Manganese availability apparently did not influence the transcript level for any isoform on day 1 (Fig. 3). Transcript levels for isoforms Fmmnp1 and Fmmnp2 displayed a similar overall pattern, which differed from Fmmnp3 levels. Transcripts of the two former isoforms increased over the time course of the experiment, peaking on day 5 in the high-manganese concentration medium, whereas Fmmnp3 levels decreased, without apparent influence by manganese concentration (Fig. 3). Isoform mnp2 transcripts were expressed at levels approximately 2-fold higher than mnp1 transcripts under any experimental treatment. On the last day of the experiment (day 7), elevated manganese levels led to decreased expression of isoforms mnp1 and mnp2, and the highest expression was recorded in the manganese-free medium.
Mean normalized expression (MNE) of mnp1, mnp2, and mnp3 transcripts from F. mediterranea grown in Mn-free medium, low-Mn concentration medium, and high-Mn concentration medium for 1, 3, 5, and 7 days.
Phylogenetic placement of the Hymenochaetales class II peroxidases.The final data set containing 177 sequences from 70 species includes 77 full-length and 100 partial sequences (see Table S1 in the supplemental material). The total length of the aligned sequences was 566 characters; 419 characters were included for phylogenetic analyses. Bayesian analysis and maximum likelihood analysis produced consensus trees with similar topologies, but results did not always agree regarding the placement of some groups lacking solid support from both analyses (results not shown). The tree shown in Fig. 2 depicts the tree with the best likelihood score (−29 188.97 ln) from the ML analysis.
The phylogenetic analysis places the majority of Hymenochaetales sequences, including the three F. mediterranea MnPs and partial sequences from Hymenochaete, Phylloporia, and Inonotus species generated by this study, at the base of clade MnP1 (Fig. 1). This clade contains the long-MnP forms, characterized by their additional disulfide bond. The remaining three partial Hymenochaetales sequences are placed in the central part of the tree (MnP3) that not only lacks support values on backbone nodes, but in this part the branching order to the tips also remains completely unresolved. The Hymenochaetales sequences do not cluster together; instead, each sequence seems to be more closely related to sequences from taxa belonging to other orders.
At the base of the tree, a small paraphyletic assemblage of basal peroxidases is placed next to the two outgroup sequences. If there is a common characteristic of this group, then it is that they seem to be monotypic; i.e., basal peroxidases do not appear in multiple isoforms. A second, albeit much larger, paraphyletic group labeled with a question mark (Fig. 1) contains sequences from white rot fungi from the Polyporales and mostly ectomycorrhizal fungi from the Agaricales and Russulales (64). The correct placement of these groups remains unresolved because the inner nodes in this part of the tree did not receive strong support values. Monophyly of all ectomycorrhizal sequences is not supported, but they are supported as members of a clade (Fig. 1, clade A) encompassing all ligninolytic peroxidases (MnP/LiP/VP). The function of the sequences in ectomycorrhizal taxa is unknown, and only limited functional data are available for the sequences from white rot taxa situated between the basal peroxidases and the core group (Fig. 1, clade A).
A large clade of MnPs (MnP1), in a sister relationship to the ectomycorrhizal sequences, receives solid support (96/1.0, bootstrap support [BS]/posterior probability [PP]) and harbors mostly sequences from the Polyporales and, branching off early, a clade (94/1.0, BS/PP) containing exclusively Hymenochaetales sequences. The central part of the tree is characterized by alternating clusters of sequences that can be attributed to MnPs and VPs. The support for these clusters is mixed. In the Agaricales, MnPs and VPs belonging to Pleurotus species are clearly separated, but an MnP from Agaricus bisporus seems to be more closely related to the VPs than to the MnPs of species of Pleurotus. The monophyly of the Pleurotus and Agaricus clade, as well as the functional separation (MnP2 and VP1), is supported only by the Bayesian analysis. A cluster containing the partial sequences from Grifola frondosa is tentatively assigned to VPs (VP2). The only full-length sequence containing all aa residues characteristic for VPs belongs to Spongipellis sp., but the position receives no support from both analyses. However, the Grifola sequences also contain the residues (tryptophan, leucine) for LiP-type substrate interaction along with the third Mn-binding residue (aspartate), which is usually missing in peroxidases without a manganese-oxidizing capacity. Support values are better for the last MnP cluster (MnP4), which contains two sets of sequences from two different Pycnoporus cinnabarinus strains. This finding is the first evidence for mnp genes in P. cinnabarinus. No differences in aa composition between the two strains are observed. Clusters MnP4 and VP2 harbor only sequences from the Polyporales. Two sequences from Bjerkandera species constitute the third and smallest VP cluster. Due to the lack of statistical support, it remains unresolved if this is indeed a third origin of VPs, but these sequences do consistently group with LiPs in phylogenetic analyses. The last major clade receives strong support from both analyses (99/1.0, BS/PP) and contains LiPs from Polyporales, including sequences from P. cinnabarinus presented here for the first time. These sequences lack the Mn-binding residues, with the exception of Trametes versicolor LP7.
The three F. mediterranea peroxidase sequences described here are the first characterized full-length sequences for this gene family from the Hymenochaetales. However, since our gene and protein characterization is based on similarities to homologous MnPs and not yet confirmed by cDNA sequences, we present these currently as predicted features only.
Despite the long evolutionary distance between the Hymenochaetales and both the Polyporales and Agaricales, which host the majority of characterized ligninolytic peroxidases, the deduced Fomitiporia protein sequences maintain a set of key characteristics that are highly conserved among fungal peroxidases. Regarding the presence of residues involved in heme pocket formation, Ca2+ binding, disulfide bond formation and length and predicted mass of the mature proteins, predicted isoelectric points, and substrate binding residues, the characterized Fomitiporia sequences adhere to the norm (2). The presence of the manganese-binding residues and the lack of the exposed tryptophan characterize the enzymes best as MnPs. Notably, there are differences compared to well-characterized MnPs. The position of the exposed tryptophan residue characterizing LiPs and VPs is usually occupied by either an alanine or serine residue in MnPs, but in the Fomitiporia MnPs this position is occupied by an asparagine (FmMnP1) or a histidine (FmMnP2 and FmMnP3). The only other class II peroxidase with a histidine in this position is from Trametes cervina (BAE46585 tclip), an enzyme that has been characterized to perform LiP-like reactions (39). This performance has been attributed to a unique surface-exposed tyrosine residue and received support from homology modeling (38, 55). However, it has also been suggested that the imidazol side group of histidine could serve as a radical stabilizing group in a manner analogous to the indol group of tryptophan (32). This would suggest a new type of VP in the Hymenochaetales, an idea that is further supported by the conservation of this residue in partial sequences of other hymenochaetalean taxa (Phylloporia ribis, Hymenochaete corrugata, Phellinidium ferrugineofuscum) that form a clade with FmMnP2 and FmMnP3. However, site-directed mutagenesis that replaced the catalytic tryptophan with a histidine in a Pleurotus eryngii VP did not support an equivalent role for histidine in this position (56). Mechanistic studies to characterize the biochemical properties of Fomitiporia peroxidases are urgently needed.
MnPs can be distinguished as long or “classical” MnPs and short MnPs (24, 41). The former contain 10 cysteine residues involved in forming five disulfide bonds, whereas the latter form only four disulfide bonds. Isoforms FmMnP1 and FmMnP2 contain 10 cysteine residues and are predicted to form five disulfide bonds. FmMnP3 is unique among all known full-length class II peroxidases in having two additional cysteine residues that could be involved in forming a sixth disulfide bond. The reduced intron number is a further characteristic that is shared between the Fomitiporia sequences and the long MnPs, but in terms of overall length the Fomitiporia sequences resemble the short MnPs more. The unique intron arrangement in Fmmnp genes could lead to further diversification in this lineage if subjected to alternative or altered splicing. The latter event has been observed in P. chrysosporium lip and mnp genes (61). However, we have no indication of the presence of additional isoforms due to aberrant transcriptional events in these genes.
We examined the expression of three mnp genes in Fomitiporia mediterranea as a function of mRNA transcript abundance. Transcript abundance can vary as a result of transcriptional activity or differences in mRNA transcript stability among the investigated genes. The expression of mnp genes in various basidiomycete species is regulated at the transcriptional level and depends on a sufficient concentration of Mn in the growth medium (7, 27, 49). Optimal Mn concentrations seem to vary among species, but up to 200 μM Mn has been found to favor expression, whereas higher concentrations can have an inhibitory effect (6, 21, 27). Even under optimal culture conditions, MnP activity tends to peak after 5 to 9 days of idiophasic growth (5, 7). Our expression results are in concordance with these findings for two of the three mnp genes. Isoform Fmmnp1 and Fmmnp2 expression levels reached a maximum at day 5 of growth in high-Mn concentration, nutrient-limited medium. Isoform Fmmnp3 transcript levels did not depend on Mn concentration, indicating a differential expression of the mnp genes in F. mediterranea. A similar observation has been made in P. chrysosporium, where the mnp3 gene is expressed in an Mn-independent way (17).
The phylogenetic analyses (Fig. 1) are consistent with our previous findings regarding the evolutionary history of the class II peroxidase family (41). These can be summarized as follows: (i) manganese peroxidases are the most widespread ligninolytic peroxidases, both in terms of taxonomic occurrence and phylogenetic distribution; (ii) VPs have evolved multiple times independently from MnP predecessors; and (iii) LiPs are monophyletic and have evolved just once in the Polyporales from a VP ancestor by losing the manganese-binding site. The addition of more sequences from previously unrepresented taxa adds further insight into the evolutionary history of the gene family. Class II peroxidases of the Hymenochaetales are not monophyletic but belong to two lineages that had separated early in the evolution of the gene family. This split distribution is also observed for sequences from other orders of the Agaricomycotina, e.g., the Polyporales and Agaricales. Presumably, an ancestral form of the Agaricomycotina already harbored multiple peroxidase genes as a result of gene duplication events before the Agaricomycotina split into its major clades.
The rather small assemblage of basal class II peroxidases lacks the residues important for ligninolysis. The only class II sequence in the recently published genome of the brown rot fungus Postia placenta belongs to this basal group supporting the notion that ligninolytic peroxidases do not play a role in brown rot decay (36).
Whether ectomycorrhizal taxa harbor ligninolytic peroxidase genes has been an intriguing question. Earlier reports could not be substantiated (10). However, a recent study showed that multiple ECM taxa harbor class II peroxidases closely related to MnPs (4). In our analysis, they cluster with the full-length sequence from Laccaria bicolor, which lacks the ligninolytic residues. Although the sequences show a higher affinity to MnPs than the basal sequences, the sister relationship to clade MnP1 shown in Fig. 1 is not strongly supported. The only ectomycorrhizal sequence that is placed with strong support within one of the ligninolytic groups is the partial sequence from Gomphus clavatus nested in clade MnP1. Our analysis suggests that this species would be a suitable candidate for further studies directed at solving the question regarding the presence of ligninolytic peroxidases in ECM fungi.
Manganese peroxidases are clearly polyphyletic. The majority of the sequences fall into one strongly supported (96/1.0) clade (Fig. 1, MnP1). The remaining groups containing MnPs (MnP2 to MnP4) are not supported as being monophyletic and are intermixed with VPs. This indicates that switches from an MnP to a VP, presumably due to a single point mutation, are not only possible but may have occurred multiple times. However, no indication of a reversal from a VP to an MnP can be drawn from the phylogenetic analysis.
Among white rot fungal taxa, P. cinnabarinus has been considered an exception because it has been reported to secrete only laccases as lignin-degrading enzymes and not peroxidases (13). Our successful amplification of peroxidase sequences and the phylogenetic analysis suggests that this fungus harbors at least three lip genes and two mnp genes. In light of these findings, there is no evidence that white rot lignin degradation is possible without any of the ligninolytic peroxidases. The sequences from the two strains we used are either identical or differ only in a single aa position. It remains to be resolved why peroxidase activity under ligninolytic conditions for P. cinnabarinus is not readily demonstrable. One possibility is that these genes are differentially regulated and are not secreted under common laboratory conditions. An alternative explanation is that the genes are expressed but not freely secreted in the surrounding medium, instead remaining attached to a sheet surrounding the fungal hyphae. A similar discrepancy between enzymatic activity and putative gene content has been observed for Phanerochaete sordida and Ceriporiopsis subvermispora, which lack LiP activity but seem to have lip genes (50, 57, 58).
This research was supported in part by National Science Foundation award DEB-0732968 to D.S.H.
The culture of F. mediterranea MF3/22 was provided as a gift by Michael Fischer. We are thankful to Inga Bödeker for providing us with class II sequence information from ectomycorrhizal taxa.
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matches: V. V. V. V. V. V. V. V.


text:
case from N.L.R.B. v. Babcock & Wilcox Co., 351 U.S. 105, 76 S.Ct.
through the usual channels . . .. Id., at 112, 76 S.Ct., at 684.
Instead, the NLRB held applicable Amalgamated Food Employees v.
in the light of Babcock. Pp. 542—548.
Norton J. Come, Washington, D.C., for respondent, N.L.R.B.
Clerk's Union 725, Retail Clerks International, AFL—CIO.
they maintain their own separate parking lots.
of the two stores which was about 80% complete.
persisted in his refusal to leave, he was arrested.
661 (1972). We conclude that it is not.
U.S., at 112, 76 S.Ct., at 684.
property rights it may require is both temporary and minimal.
Local 590 v. Logan Valley Plaza, Inc., 391 U.S. 308, 88 S.Ct.
constitutional grounds; it is not a § 7 case.
Logan Valley had its genesis in Marsh v. Alabama, 326 U.S.
320, 88 S.Ct., at 1609.
trespass laws of the State against the pickets.
functional attributes of public property devoted to public use.
the Court of Appeals erred in applying Logan Valley to this case.
because it viewed Logan Valley as controlling rather than Babcock.
to that court for such consideration.
djQ Mr. Justice MARSHALL, with whom Mr. Justice DOUGLAS and Mr.
may communicate with employees on the property of the employer.
employees. 439 F.2d 1321 (1971).
U.S. 156, 168, 83 S.Ct. 239, 245, 9 L.Ed.2d 207 (1962).
further proceedings without deciding the constitutional question.
See Thomas v. Collins, 323 U.S. 516, 533—534, 65 S.Ct.
315, 324—325, 89 L.Ed. 430 (1945).
Babcock & Wilcox Co., 109 N.L.R.B. 485, 486 (1954).3 NLRB v. Babcock & Wilcox Co., 222 F.2d 316 (CA5 1955).
Brief for the NLRB 20.5 439 F.2d, at 1326—1328.

matches:
v.
v.
v.
§ 7
v.
v.
v.


Elizabeth Manos for the plaintiff.
Shahan J. Kapitanyan for the defendant.
GRECO, P.J. Advanced Spine Centers, Inc. (Advanced Spine) brought this suit against Commerce Insurance Company (Commerce) seeking Personal Injury Protection (PIP) benefits in the amount of $3,240.00, which represented its bills for chiropractic services rendered to Noun Meas (Meas). The complaint alleged that Meas was a passenger injured in an accident while in a vehicle insured by Commerce. After a jury trial, judgment was entered for Advanced Spine in the above amount, along with attorneys fees in the amount of $13,925.94. On this appeal, Commerce argues that the trial judge erred in (1) relieving [Advanced Spine] from presenting affirmative evidence at trial to meet its burden of proof, (2) allowing into evidence medical records submitted under G.L. c. 233, § 79G by way of an affidavit of its keeper of records, (3) refusing to provide to the jury a missing witness instruction, (4) denying its request to enter into the record subpoenas served on the alleged missing witness, and (5) awarding Advanced Spines attorney excessive and unreasonable fees. We discuss these issues in the context of how they arose in the trial court.
Pursuant to G.L. c. 233, § 79G, medical bills and reports that are subscribed and sworn to under the penalties of perjury by the physician . . . rendering such services . . . shall be admissible as evidence of the fair and reasonable charge for such services or the necessity of such services or treatments. Under the statute, a chiropractor is considered a physician. Id. See also Mass. G. Evid. § 803(6)(C)(i)(b), at 254 (2012). In Ortiz v. Stein, 31 Mass. App. Ct. 643 (1991), the Appeals Court rejected the argument that the scope of § 79G [was] sufficient to include the opinion report of a physician as to proximate cause without regard to whether the physician treated or examined the person claiming injury, stating that such a position was an inventive interpretation [that] places impossible strain upon the plain language of § 79G . . . [and] also runs counter to the purpose of that statute. Id. at 645. In the case at bar, there is no indication that the affiant, Kohn, was a physician or a chiropractor. He identified himself only as a keeper of the records. In these circumstances, Commerce maintains that he had no personal knowledge of the necessity of the treatment, thereby rendering the attached records inadmissible hearsay. However, we see nothing in § 79G that would preclude a keeper of the records from collecting various records that individually contained the requisite attestations by the treating chiropractor and submitting them as one document.
2. Evidence at trial. The plaintiff, Advanced Spine, presented no witnesses, but relied exclusively on Commerces answers to interrogatories and its medical records. As to the answers, Commerce admitted that it was the automobile insurance carrier providing . . . [PIP] coverage . . . for the injured person [Meas] with regard to the accident alleged in [Advanced Spines] complaint; that it was notified of the accident on or about June 28, 2008; that Advanced Spine submitted bills in the amount of $4,835.00 for services it rendered to Meas; and that, of that amount, Commerce paid only $1,595.00. The only other evidence presented by Advanced Spine were medical records referred to above. With that, Advanced Spine rested. Commerce then moved for a directed verdict, which was denied.
In response, Commerce presented two witnesses. Zing Ding testified that she was the adjuster assigned to this claim. When she received Advanced Spines bills, she referred them to Dr. Stephen Leonti (Leonti) for review. Leonti testified that, in his opinion, based on his review of Advanced Spines records and other information he received, the treatment rendered by Advanced Spine through August 14, 2008 would have been customary and reasonable for a man of Meas age who basically sustained uncomplicated muscular strains related to the vehicular incident at issue in this case. Thus, Leonti concluded that Commerces prior payment of only $1,595.00 was justified.
Spine was rather sparse, it did cover these elements. Answers to interrogatories, if properly admitted at trial, are evidentiary admissions that may be considered by the fact finder. Id. at 48. See also General Elec. Co. v. Board of Assessors of Lynn, 393 Mass. 591 , 603 n.8 (1984) (Answers to interrogatories in the same case constitute evidentiary, not judicial, admissions.). In this case, Commerces answers supplied evidence of the first three elements noted above. A further inference as to those elements can be drawn from Commerces payment of $1,595.00 of the bills. Moreover, the substance of Commerces answers would have been based on its own institutional knowledge. Finally, the medical records were evidence, whether believed or not, that all the treatment was reasonable and necessary and that Advanced Spine was due the remaining amount sought. In these circumstances, the court did not err in denying Commerces motions for a directed verdict, both after Advanced Spine presented its evidence and after the close of all the evidence.
3. Missing witness. Before closing arguments, Commerce sought a missing witness instruction applicable to Culliney. The motion was denied, apparently without any discussion at that point. [Note 1] The instruction was not given. However, the prospect of Cullineys testimony was discussed at length before the trial began. Culliney was one of two chiropractors who treated Meas. Patel initially treated him for a short period, from July 9, 2008 to July 24, 2008. All of the bills generated by Patel were within the $1,595.00 paid by Commerce. Thereafter, until December 15, 2008, Culliney treated Meas. Thus, all the challenged bills related to Cullineys treatment. At the pretrial discussion, Commerces attorney recounted his unsuccessful efforts to serve Culliney with a subpoena, [Note 2] thereby limiting his right, as provided in G.L. c. 233, § 79G, to cross-examine Culliney with respect to such bill, record and report or to rebut the contents thereof. Advanced Spines attorney stated that Culliney was no longer employed by Advanced Spine, and that he may be living in Kentucky. Commerce also argued that Advanced Spines attorney should be able to accept the subpoena since he was representing Culliney in an unrelated civil action in the Federal Court.
Mass. G. Evid. §1111, at 370 (2012). In this case, neither party appeared to know where Culliney could be found. The trial judge could have also considered that Commerce was able to present evidence concerning the necessity of the treatment through its witness, Leonti. Thus, Commerce had a live witness who testified that the treatment at issue here was not necessary. In these circumstances, it cannot be said that the trial judge abused his discretion in not giving the instruction sought, or in not admitting the subpoenas into evidence.
Neither party objected to the charge. Nor was there an objection to the verdict slip presented to the jury, which asked the question, Did Advanced Spine prove that Commerce failed to pay all of its reasonable medical bills for necessary chiropractic treatment rendered to Noun Meas? While the judge did not list the elements as done in Provenzano, the issues were clear. There was no shifting of the burden of proof.
5. Attorneys fees. After trial, Advanced Spines counsel sought attorneys fees in the amount of $13,662.50 [Note 3] for services performed by himself and an associate. Commerce argued that the fee was excessive primarily in view of the amount recovered, to wit, $3,240.00. It did not quarrel with the hourly rate charged by the two attorneys. The amount of damages involved [and] the result obtained are only two of several factors a judge must consider. Linthicum v. Archambault, 379 Mass. 381 , 389 (1979).
Under all the Linthicum factors, we cannot say that the trial judge abused his discretion in making his award. However, we remand this matter to the trial court for clarification and possible correction. Exhibit A attached to counsels affidavit indicates that he is seeking compensation for two separate eight-hour periods for the court appearances of his associate on August 4, 2010. If, in fact, this was a duplication, the fee award would have to be reduced by $2,000.00.
Accordingly, other than the above clarification, the judgment of the trial court is affirmed.
[Note 1] The trial judge also denied Commerces motions to hold Culliney in contempt; to issue a capias for his arrest, or in the alternative, to continue the trial; to admit into evidence copies of subpoenas sent to Culliney; and to require Culliney to appear at the hearing related to the G.L. c. 93A count.
[Note 2] One of the subpoenas was left with Cullineys wife, another received by his son for his father. However, there was no evidence that Culliney was living with his family at the time.
[Note 3] The amount was ultimately increased to $13,925.94.


A TUMBLING-T RANCHES, an Arizona general partnership; RUSSELL BADLEY FARMS, INC., an Arizona corporation; ROSEMARY L. EDWARDS, individually and as Trustee of the Rosemary L. Edwards Trust; JOHN E. FORNES, Jr. and SHELLEY FORNES, husband and wife; PJ FARMS LIMITED PARTNERSHIP, an Arizona limited partnership; J A FORNES, II, an Arizona general partnership; MARION GETZWILLER and POLLY GETZWILLER, husband and wife; DELMAR JOHN and JEAN JOHN, husband and wife, d/b/a DELMAR JOHN FARMS; BDJ FARMS, L.L.C., an Arizona limited liability company; GILA RIVER FARMS, INC., an Arizona corporation; ROY PIERPOINT and ELLA PIERPOINT, husband and wife; PIERPOINT FARMS, INC., an Arizona corporation; and WOOD BROTHERS FARMS, an Arizona general partnership, Plaintiffs-Appellants, v. PALOMA INVESTMENT LIMITED PARTNERSHIP, a limited partnership; PRUDENTIAL INSURANCE COMPANY OF AMERICA, a New Jersey corporation; MARICOPA LAND, INC., an Arizona corporation; PALOMA RANCH JOINT VENTURE, a joint venture; BOOKMAN EDMONSTON ENGINEERING, INC., a California corporation; MARICOPA COUNTY FLOOD CONTROL DISTRICT, a body politic; MARICOPA COUNTY, a body politic, Defendants-Appellees.
1 CA-CV 98-0605.Court of Appeals of Arizona, Division One, Department E.
Appeal from the Superior Court in Maricopa County, Cause Nos. CV 95-00253 and CV 95-00290 (Consolidated).
Jennings, Strouss Salmon, P.L.C., Phoenix, By John J. Egbert, and Kinerk, Beal, Schmidt Dyer, P.C., Tucson, By Burton J. Kinerk and Law Offices of Reed King, Phoenix, By Reed King, Attorneys for Plaintiffs-Appellants.
Helm Kyle, Ltd., Tempe, By John D. Helm, Sarah A. Worthington, and Lorrie L. Luellig, Attorneys for Defendant-Appellee Maricopa County Flood Control District.
Teilborg, Sanders Parks, Phoenix, By John C. Gemmill and Kendall D. Steele, Attorneys for Defendants-Appellees Prudential Insurance Co. of America and Paloma Ranch Joint Venture.
Mesch, Clark Rothschild, P.C., Tucson, By J. Emery Barker and Scott H. Gan, Attorneys for Defendant-Appellee Paloma Investment Limited Partnership.
¶ 1 Appellants, owners of farms located along the Gila River, brought this action to recover for damages they suffered during the 1993 flooding of the river. Appellants alleged that Paloma Investment Limited Partnership, Prudential Insurance Company of America, Maricopa Land, Inc., Paloma Ranch Joint Venture, Bookman-Edmonston Engineering, Inc., and Maricopa County, by virtue of their maintenance, ownership, and control of Gillespie Dam and adjacent land and stream beds, and the Maricopa County Flood Control District (“FCD”), by virtue of its construction of an upstream clearing and pilot channel project, caused water and sediment to be diverted, obstructed, or rerouted through Appellants’ properties, resulting in significant damage to real and personal property. Appellants appeal from the trial court’s grant of summary judgment in favor of Appellees. We have appellate jurisdiction pursuant to Arizona Revised Statutes Annotated section 12-2101(B) (1994).
I. ¶ 2 We view the facts and all reasonable inferences therefrom in the light most favorable to the parties opposing summary judgment. See Orme Sch. v. Reeves, 166 Ariz. 301, 309-10, 802 P.2d 1000, 1008-09 (1990). Gillespie Dam is a concrete and steel structure approximately 1700 feet long and 20 feet high. It was constructed on the Gila River in 1921 as an irrigation diversion dam. Appellants’ properties lie along a thirty-seven mile stretch of the river, extending from a point approximately one and one-half miles below the dam on the north to the Painted Rock Reservoir on the south.
¶ 3 Gillespie Dam’s reservoir filled with silt and sediment up to its crest shortly after it was completed. The sediment formed a triangular wedge approximately 1700 feet wide and 20 feet deep at the face of the dam that tapered off five or six miles upstream. The dam and sediment wedge remained intact until the 1993 flood. Several Appellants attested that prior floods in 1978 and 1980, one of which set a record for peak water flow, caused little or no damage to their farms.
¶ 4 Property owners upstream, however, had experienced flooding problems. In response, FCD embarked on two flood control projects. In 1979, FCD cleared vegetation from a 1000-foot-wide corridor running from 91st Avenue to the dam in order to improve channel flow and thus prevent high flows of water from being forced onto adjacent farmland. In 1986, FCD began constructing a “pilot channel” upstream from the dam. The channel, which was intended to straighten and direct water flow in that portion of the river, averaged three feet deep, fifty to one hundred feet wide, and extended over twenty-two miles. FCD completed both projects in 1992.
¶ 5 In early 1993, the Gila watershed experienced record rainfall. Flooding occurred from January 1 through April 28, 1993, setting records for runoff volume and duration of flooding. On January 9, 1993, the river reached a high peak flow between 7:00 a.m. and 11:00 a.m., which was the longest duration peak flow in the river’s recorded history. Several hours later, the dam incurred an initial break approximately twenty to twenty-five feet wide, which ultimately widened to 206 feet. The water rushed through the breach, creating a headcut, or trench, in the sediment wedge. This headcutting process continued for several months thereafter and extended for several miles upstream. Appellants’ experts estimated that thirty-six million cubic yards of additional sediment were introduced into the river by the dam’s breach and the ensuing erosion of the sediment wedge.
¶ 6 Appellants brought suit against Appellees, alleging that Appellees’ conduct relating to the breach of the dam and the clearing and pilot projects caused the release of this additional sediment, which clogged the channel, caused more water to overflow the banks, and thus caused increased damage to Appellants’ properties. Appellants also alleged that the sediment deposits increased the risk of damage to their properties from future flooding. Appellants based their claims on theories of negligence, strict liability, trespass, nuisance, and in the case of Maricopa County and FCD, inverse eminent domain.
¶ 8 In these motions, Appellees contended that Appellants suffered no damage due to the breach and that the alleged damage was due to Appellants’ encroachment on the river’s floodplain and the “extensive and sustained natural flooding.” Appellees relied upon the opinions of at least ten experts in preparing their motions and supporting statements of facts.
1. After the breach of the dam 36,000,000 more cubic [yards] of silt and sediment flowed down river.
2. Some undetermined amount of that silt and sediment was deposited in the river bed and on the Plaintiffs’ properties. The remainder was deposited in the Painted Rock Reservoir.
3. As a result of that increased deposit, the Plaintiffs’ properties are at some greater risk for flooding in the future.
A jury could not find from the evidence that the breach of the dam increased the erosion to the Plaintiffs’ properties during the floods; nor that the breach increased the loss of various fixtures, fencing, wells or berms.
The evidence, as presented by the Plaintiffs, upon which the jury could consider how much the possibility of flooding has increased as a result of the silt and sediment deposits as to all of the Plaintiffs; and how any individual Plaintiff has been damaged, is insufficient to meet the standards of Orme School. . . .
The trial court then granted Appellees’ motions for summary judgment.
¶ 10 Appellants subsequently filed a motion for new trial. In this motion, Appellants argued that the Schlecht case had long been superseded by the development of the “single injury” rule of Holtz v. Holder, 101 Ariz. 247, 251, 418 P.2d 584, 588 (1966), and that under this rule, defendants, not plaintiffs, bear the burden of proving what portion of the harm was caused by their conduct. Appellants further argued that on the “any increase in damage” issue, the trial court’s findings that Appellants suffered “some” damage was enough to defeat Appellees’ summary judgment motions.
consistent with the holding of Holtz v. Holder. . . . [H]owever, the plaintiffs still must demonstrate that the defendants’ actions or inaction contributed to their damage, that is that the injury was increased by the defendants’ action or inaction. . . . [O]nly then, does the burden shift to the defendants to prove who bears what responsibility.
Here all of the damages before and after the breach of the dam were of the same type, the plaintiff’s mission was to show that by the breach in the dam, the harm to the plaintiffs was increased. The Court’s review of this matter does not persuade the Court that the plaintiffs have carried that burden.
The trial court denied Appellants’ new trial motion.
II. ¶ 12 In an appeal from summary judgment, we “determine de novo whether genuine issues of material fact exist and whether the trial court correctly applied the law.” Floyd v. Donahue, 186 Ariz. 409, 411, 923 P.2d 875, 877 (App. 1996). A motion for summary judgment “should be granted if the facts produced in support of the claim or defense have so little probative value, given the quantum of evidence required, that reasonable people could not agree with the conclusion advanced by the proponent of the claim or defense.” Orme School, 166 Ariz. at 309, 802 P.2d at 1008. “Where reasonable minds could reach different conclusions as to the existence of a genuine issue, summary judgment should not be granted.” Tribe v. Shell Oil Co., 133 Ariz. 517, 518, 652 P.2d 1040, 1041 (1982).
(App. 1978) (citing Restatement (Second) of Torts, § 432(1), illus. 2 (1965)). But if Appellees’ allegedly negligent acts were a cause of “some part” of Appellants’ damages, Appellees would be liable for at least the damages they caused. Id.
the canal and did not result from the escape of water through certain breaches as the homeowners maintained. See id. at 338, 576 P.2d at 526. At the close of evidence, the trial court granted a directed verdict in favor of the Association. See id. at 333, 576 P.2d at 521. On appeal, this court acknowledged that the water overtopping the canal could have caused part of the damages; however, it concluded that “a jury could find that the flood waters escaping the canal [through the breaches] possessed greater volume and greater kinetic force than they would have had absent the Association’s negligence, and that this contributed to appellants’ injuries.” Id. at 339, 576 P.2d at 527.
As in Markiewicz, a jury could conclude that more water flowing at a greater rate contributed to appellants’ damages. Therefore, in our opinion, there is sufficient evidence of causation in the record to preclude summary judgment on causation.
Taft, 169 Ariz. at 177-78, 818 P.2d at 162-63. Hence, under Markiewicz and Taft, to defeat a motion for summary judgment, a plaintiff must produce evidence that the defendant caused “some part” of the plaintiff’s damages, which need not be quantified or apportioned. Markiewicz, 118 Ariz. at 338, 576 P.2d at 526; Taft, 169 Ariz. at 177, 818 P.2d at 162. The record here reveals that Appellants met this test.
¶ 16 In response to the motions for summary judgment, Appellants submitted the affidavit testimony of Dr. Daryl B. Simons. Dr. Simons holds a Ph.D. degree in hydraulics and river mechanics from Colorado State University and is a registered professional engineer in Arizona and several other states. He has served as an Associate Dean of the Colorado State University College of Engineering and has written several textbooks on sediment transport technology and fluid mechanics. Dr. Simons explained that FCD’s pilot channel project increased the average gradient of the river channel above the dam. The clearing project removed vegetation that previously had held sediment in place and slowed water flow in that stretch of the river. He stated that both of these projects increased water velocity during the 1993 flood, which caused the river to pick up and carry downstream substantially more sediment than would have been carried by the natural channel.
¶ 17 Dr. Simons explained that when the dam broke, the water poured through the breach, which increased the water’s ability to carry more silt and sediment from the upstream portions of the river through the breach and into the river’s lower reach. The high-velocity water created a headcut in the sediment wedge, and forty-one million cubic yards of sediment were released from the river bed above the dam and carried to the area between the dam and Painted Rock Reservoir. Much of that sediment was deposited in and along Appellants’ properties. Dr. Simons attested that had the dam not breached, computer analysis demonstrated that only five million cubic yards of silt and sediment would have passed over the dam. The breach thus caused an additional thirty-six million cubic yards of sediment to be deposited in the lower reach of the river, according to Dr. Simons.
¶ 18 Dr. Simons further explained that the great volume of sediment created new sandbars and islands, obstructed the channel, and changed the direction of the floodwater flows. Water that otherwise would have flowed in the channel was diverted onto farmlands, increasing erosion, crop loss, and loss of wells, ditches, and other fixtures. Dr. Simons attested that the sediment reduces the river’s ability to carry future floodwaters and thus increases the risk of property damage from future floods.
¶ 19 Appellants also submitted the affidavit of Dr. William L. Graf. Dr. Graf holds a Ph.D. in physical geography and water resources management from the University of Wisconsin. Currently a professor at Arizona State University, he specializes in fluvial geomorphology, hydrology, and aerial photograph interpretation. Rather than calculating the amount of sediment deposited on Appellants’ land as Dr. Simons had done, Dr. Graf based his opinions on personal observations of the riverbed before and after the flood, on the testing of soil samples, and on analysis of aerial photographs. Dr. Graf observed that the once-open river channel had been covered by significant quantities of sediment. He observed significant erosion of the banks and perimeter of the channel caused by water that normally would have occupied the channel but for the sediment that now filled it. Dr. Graf’s soil analysis confirmed that much of this sediment filling the channel came from the upstream portion of the dam. Dr. Graf concurred with Dr. Simons’ opinion that the FCD’s pilot and clearing projects and the breach of the dam caused substantial amounts of sediment to be deposited in the river channel adjacent to Appellants’ properties, directly contributing to Appellants’ damage. We conclude that the opinions proffered by Dr. Simons and Dr. Graf created a prima facie case for Appellants.
carried through the breach, and deposited on or near their properties than would have been carried over the dam and deposited absent the breach and the pilot and clearing projects. This evidence creates a genuine issue of fact regarding whether the increased volume of sediment contributed to Appellants’ damages. See Markiewicz, 118 Ariz. at 339, 576 P.2d at 527; Taft, 169 Ariz. at 178, 818 P.2d at 163. Because the record supports an inference that Appellees’ conduct contributed to Appellants’ damages, we reverse the trial court’s grant of summary judgment to Appellees.
¶ 23 Furthermore, we agree with Appellants that in a case such as this, where multiple tortfeasors cause indivisible damage, the Schlecht case has long been superseded by the single injury rule of Holtz and its progeny. In Schlecht, the plaintiff suffered damage after the defendant constructed a wall in a wash that diverted stream water onto the plaintiff’s property during a rainstorm. 76 Ariz. at 217, 262 P.2d at 254. The supreme court approved a jury instruction that stated that it was “not sufficient” for a plaintiff to show that a defendant caused some damage, but that the plaintiff must show “how much of the damage” was caused by the defendant or encounter a defense verdict. Id. at 219-20, 262 P.2d at 255. Later, in Holtz, the supreme court adopted the “single injury” rule where a plaintiff suffered indivisible injury from separate accidents. 101 Ariz. at 248, 251, 418 P.2d at 585, 588. There, the court shifted the burden of apportionment to the defendants, noting that “it is more desirable, as a matter of policy, for an injured and innocent plaintiff to recover his entire damages jointly and severally from independent tortfeasors . . . than it is to let two or more wrongdoers escape liability altogether, simply because the plaintiff cannot carry the impossible burden of proving the respective shares of causation.” Id. at 251, 418 P.2d at 588.
¶ 24 Recently, in Piner v. Superior Court, 192 Ariz. 182, 962 P.2d 909 (1998), the supreme court considered the status of the single injury rule in light of the legislature’s abolition of joint and several liability. The court held that “the present version of UCATA has left intact the rule of indivisible injury, relieving the plaintiff of apportioning damage according to causal contribution.” Id. at 188, ¶ 26, 962 P.2d at 915. The court noted that this rule applies to both multiple causation and successive injury cases: “Like our predecessors in Holtz, we see no reason to employ a different rule if the injuries occur at once, five minutes apart or, as in the present case, several hours apart. The operative fact is simply that the conduct of each defendant was a cause and the result is indivisible damage.” Id. at 189, ¶ 27, 962 P.2d at 916.
¶ 25 Thus, once a plaintiff proves that the defendants’ conduct contributed to the plaintiff’s damages, see Markiewicz, 118 Ariz. at 338, 576 P.2d at 526, the burden of proof shifts to the defendants to apportion damages. See Piner, 192 Ariz. at 189, ¶¶ 28, 30, 962 P.2d at 916; Holtz, 101 Ariz. at 251, 418 P.2d at 588.
¶ 27 Because of our resolution of the summary judgment issue, the issue raised by Appellants regarding Appellees’ purported “Act of God” defense is moot. We therefore do not address it.
their properties beyond that due to the natural flood itself. This evidence satisfies the requirements of Markiewicz and Taft, and it creates a genuine issue of fact regarding whether Appellees caused any increase in Appellants’ damages. Accordingly, we reverse the trial court’s grant of summary judgment to Appellees and remand the case for further proceedings.
 Maricopa County and Bookman-Edmonston Engineering, Inc. were dismissed from this lawsuit.