First report of Colletotrichum shisoi causing anthracnose of Perilla frutescens in the United States.

Perilla mint (Perilla frutescens (L.) Britt.) is an annual plant native to Asia and considered invasive in North America where it has escaped cultivation as an ornamental (Miller 1947; Swearingen et al. 2010). In August 2021, an anthracnose disease was observed on invasive perilla found along the disturbed margins of a forest in Frederick County, Maryland, United States. Symptoms included necrotic lesions with chlorotic halos, were concentrated in the lower canopy, and caused premature defoliation of lower leaves (Figure S1). Leaves from four plants were surface sterilized by rinsing for 30 s in 70% ethanol, 60 s in 0.8% NaClO, and 60 s in sterile water and then incubated on 2% water agar under ambient laboratory conditions to permit sporulation. After three days, spores that exuded from individual lesions were streaked onto acidified potato dextrose agar. Two single-conidium isolates were recovered from each plant. All eight isolates were identified to species using DNA sequences. A single isolate (21-067) was selected at random for morphological characterization and completion of Koch's postulates. Morphological features were recorded after seven days of growth on synthetic low-nutrient agar (SNA) and potato dextrose agar (PDA) incubated at 22°C under 12 hr UV-B and white fluorescent lighting. Measurements were based on a minimum of 20 observations per structure. Cultures on SNA were flat, hyaline to pale salmon, lacked sporodochia and grew at a rate of 1.3 mm day-1 (n = 3). Vegetative hyphal width was (minimum-maximum) 1.5-4.0 µm, (average ± standard deviation) 2.7 ± 0.9 µm. Conidiophores arose directly from hyphae, were hyaline, smooth, unbranched and measured 40.0-180.0 x 1.5-4.5 µm, 102.3 ± 33.9 x 2.7 ± 0.8 µm. Conidia were single celled, straight, hyaline, glabrous, rounded at both ends and measured 10.0-20.0 x 3.8-6.3 µm, 15.4 ± 2.5 x 4.9 ± 0.7 µm. Setae, observed only on PDA, were pale brown, 1-2 septate, straight, blunt tipped and measured 42.5-97.5 µm, 70.8 ± 13.4 µm. Appressoria formed on PDA were single-celled, pigmented, smooth, and obovoid with entire margins, measuring 5.2-7.7 x 8.6-13.8 µm, 6.8 ± 0.6 x 10.8 ± 1.4 µm. These characteristics were consistent with members of the Colletotrichum destructivum species complex. Partial DNA sequences from five loci were amplified following the procedures of Damm et al. 2014: internal transcribed spacer region of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and beta-tubulin (TUB2). Pairwise sequence comparisons to references using the Blastn algorithm found 99.8% similarity between isolate 21-067 and ex-type C. shisoi isolate JCM 31818: MH660930 (ITS, 545/546 bp), MH660931 (GAPDH,192/192 bp), MH660929 (CHS-1, 278/280 bp), MH660928 (ACT, 262/262 bp), and MH660932 (TUB2, 511/511 bp) (Altschul et al. 1990; Gan et al. 2019). Subsequently, sequences from all eight isolates were aligned with Clustal Omega (Sievers and Higgins 2018), concatenated, and used in a Bayesian phylogenetic reconstruction (Huelsenbeck and Ronquist 2001) of the C. destructivum species complex (Figure S2), confirming the species identity as C. shisoi. Sequences were deposited in NCBI GenBank under accession numbers OM865277-OM865284 and OM885059-OM885090. Koch's postulates were fulfilled using perilla grown in a greenhouse until second true leaves emerged. Inoculum washed from two-week-old fungal cultures grown on potato dextrose agar was adjusted to 4 x 104 conidia mL-1 and applied to three replicate plants with an aspirator until runoff. Three plants were sprayed with sterile water as a negative control. Plants were covered with plastic bags for 72 hrs and maintained in a growth chamber at 20°C and 80% RH for 14 days. Inoculated plants displayed disease symptoms similar to those observed under field conditions, and control plants did not develop symptoms. Colletotrichum shisoi was reisolated from symptomatic tissue and reidentified based on morphology. The experiment was completed twice. To the authors' knowledge, this is the first report of C. shisoi on P. frutescens in the United States. Colletotrichum shisoi has not been reported as a pathogen on other plants in the United States and may have potential use as a biological control agent for invasive perilla.

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858).

UNLABELLED: AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two ß-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. IMPORTANCE: Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal ß-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.

The ABC-F protein EttA gates ribosome entry into the translation elongation cycle.

ABC-F proteins have evaded functional characterization even though they compose one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein energy-dependent translational throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistently with this inference, EttA-deleted cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a new mechanism sensitive to cellular energy status.

The impact of surgeon choice on the cost of performing laparoscopic appendectomy.

INTRODUCTION: While laparoscopic appendectomy (LA) can be performed using a myriad of techniques, the cost of each method varies. The purpose of this study is to analyze the effects of surgeon choice of technique on the cost of key steps in LA. METHODS: Surgeon operative notes, hospital invoice lists, and surgeon instrumentation preference sheets were obtained for all LA cases in 2008 at Cambridge Health Alliance (CHA). Only cases (N = 89) performed by fulltime staff general surgeons (N = 8) were analyzed. Disposable costs were calculated for the following components of LA: port access, mesoappendix division, and management of the appendiceal stump. The actual cost of each disposable was determined based on the hospital's materials management database. Actual hospital reimbursements for LA in 2008 were obtained for all payers and compared with the disposable cost per case. RESULTS: Disposable cost per case for the three portions analyzed for 126 theoretical models were calculated and found to range from US $81 to US $873. The surgeon with the most cost-effective preferred method (US $299) utilized one multi-use endoscopic clip applier for mesoappendix division, two commercially available pretied loops for management of the appendiceal stump, and three 5-mm trocars as their preferred technique. The surgeon with the least cost-effective preferred method (US $552) utilized two staple firings for mesoappendix division, one staple firing for management of the appendiceal stump, and 12/5/10-mm trocars for access. The two main payers for LA patients were Medicaid and Health Safety Net, whose total hospital reimbursements ranged from US $264 to US $504 and from US $0 to US $545 per case, respectively, for patients discharged on day 1. DISCUSSION: Disposable costs frequently exceeded hospital reimbursements. Currently, there is no scientific literature that clearly illustrates a superior surgical method for performing these portions of LA in routine cases. This study suggests that surgeons should review the cost implications of their practice and to find ways to provide the most cost-effective care without jeopardizing clinical outcome.

Coordinated clinical and financial analysis as a powerful tool to influence vendor pricing.

BACKGROUND: As costs continue to outpace reimbursements, hospital administrators and clinicians face increasing pressure to justify new capital purchases. Massachusetts Health Care Reform has added further economic challenges for Disproportionate Share Hospitals (DSH), as resources formerly available to treat the uninsured have been redirected. In this challenging climate, many hospitals still lack a standardized process for technology planning and/or vendor negotiation. PURPOSE: : The purpose of this study was to determine whether a simple, coordinated clinical and financial analysis of a technology, Endoscopic Carpal Tunnel Release (ECTR), is sufficient to impact vendor pricing at Cambridge Health Alliance (CHA), a disproportionate share hospital (DSH) in Cambridge, Massachusetts. METHODOLOGY: This case study addressed the topic of technology adoption, a complex decision-making process every hospital administration faces. Taking note of other hospitals approaches to instill a strategic management culture, CHA combined a literature review on clinical outcomes and financial analysis on profitability. Clinical effectiveness was evaluated through a literature review. The financial analysis was based on a retrospective inquiry of fixed and variable costs, reimbursement rates, actual payer mix, and profitability of adopting ECTR over open carpal tunnel release at CHA. This clinical and financial analysis was then shared with the vendor. FINDINGS: A literature review revealed that although there are short-term benefits to ECTR, there is little to no difference in long-term outcomes to justify a calculated incremental loss of $91.49 in revenue per case. Sharing this analysis with the vendor resulted in a 30% price reduction. A revised cost analysis demonstrated a $53.51 incremental gain in revenue per case. CHA has since elected to offer ECTR to its patients. PRACTICE IMPLICATIONS: Smaller hospital systems often have modest leverage in vendor negotiations. Our results suggest that the development of adoption criteria and an evidence-based managerial approach can create dialogue with vendors and directly impact pricing. Coordinated clinical and financial analysis is a powerful tool, enabling administrators, clinicians, and medical device suppliers to work constructively to provide patients access to innovative technology, even in the face of a challenging payer mix. Ongoing assessment of clinical outcomes and financial data must be performed to reflect the most up-to-date scientific and economic climate.

Phospholipid-induced monomerization and signal-peptide-induced oligomerization of SecA.

The SecA ATPase drives the processive translocation of the N terminus of secreted proteins through the cytoplasmic membrane in eubacteria via cycles of binding and release from the SecYEG translocon coupled to ATP turnover. SecA forms a physiological dimer with a dissociation constant that has previously been shown to vary with temperature and ionic strength. We now present data showing that the oligomeric state of SecA in solution is altered by ligands that it interacts with during protein translocation. Analytical ultracentrifugation, chemical cross-linking, and fluorescence anisotropy measurements show that the physiological dimer of SecA is monomerized by long-chain phospholipid analogues. Addition of wild-type but not mutant signal sequence peptide to these SecA monomers redimerizes the protein. Physiological dimers of SecA do not change their oligomeric state when they bind signal sequence peptide in the compact, low temperature conformational state but polymerize when they bind the peptide in the domain-dissociated, high-temperature conformational state that interacts with SecYEG. This last result shows that, at least under some conditions, signal peptide interactions drive formation of new intermolecular contacts distinct from those stabilizing the physiological dimer. The observations that signal peptides promote conformationally specific oligomerization of SecA while phospholipids promote subunit dissociation suggest that the oligomeric state of SecA could change dynamically during the protein translocation reaction. Cycles of SecA subunit recruitment and dissociation could potentially be employed to achieve processivity in polypeptide transport.

Crystal structures of the BtuF periplasmic-binding protein for vitamin B12 suggest a functionally important reduction in protein mobility upon ligand binding.

BtuF is the periplasmic binding protein (PBP) for the vitamin B12 transporter BtuCD, a member of the ATP-binding cassette (ABC) transporter superfamily of transmembrane pumps. We have determined crystal structures of Escherichia coli BtuF in the apo state at 3.0 A resolution and with vitamin B12 bound at 2.0 A resolution. The structure of BtuF is similar to that of the FhuD and TroA PBPs and is composed of two alpha/beta domains linked by a rigid alpha-helix. B12 is bound in the "base-on" or vitamin conformation in a wide acidic cleft located between these domains. The C-terminal domain shares structural homology to a B12-binding domain found in a variety of enzymes. The same surface of this domain interacts with opposite surfaces of B12 when comparing ligand-bound structures of BtuF and the homologous enzymes, a change that is probably caused by the obstruction of the face that typically interacts with this domain by the base-on conformation of vitamin B12 bound to BtuF. There is no apparent pseudo-symmetry in the surface properties of the BtuF domains flanking its B12 binding site even though the presumed transport site in the previously reported crystal structure of BtuCD is located in an intersubunit interface with 2-fold symmetry. Unwinding of an alpha-helix in the C-terminal domain of BtuF appears to be part of conformational change involving a general increase in the mobility of this domain in the apo structure compared with the B12-bound structure. As this helix is located on the surface likely to interact with BtuC, unwinding of the helix upon binding to BtuC could play a role in triggering release of B12 into the transport cavity. Furthermore, the high mobility of this domain in free BtuF could provide an entropic driving force for the subsequent release of BtuF required to complete the transport cycle.

ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer.

It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by the E171Q mutant of the MJ0796 ABC, which is hydrolytically inactive due to mutation of the catalytic base. The structure shows a symmetrical dimer in which two ATP molecules are each sandwiched between the Walker A motif in one subunit and the LSGGQ signature motif in the other subunit. These results establish the stereochemical basis of the power stroke of ABC transporter pumps.

The crystal structure of the olfactory marker protein at 2.3 A resolution.

Olfactory marker protein (OMP) is a highly expressed and phylogenetically conserved cytoplasmic protein of unknown function found almost exclusively in mature olfactory sensory neurons. Electrophysiological studies of olfactory epithelia in OMP knock-out mice show strongly retarded recovery following odorant stimulation leading to an impaired response to pulsed odor stimulation. Although these studies show that OMP is a modulator of the olfactory signal-transduction cascade, its biochemical role is not established. In order to facilitate further studies on the molecular function of OMP, its crystal structure has been determined at 2.3 A resolution using multiwavelength anomalous diffraction experiments on selenium-labeled protein. OMP is observed to form a modified beta-clamshell structure with eight antiparallel beta-strands. While OMP has no significant sequence homology to proteins of known structure, it has a similar fold to a domain found in a variety of existing structures, including in a large family of viral capsid proteins. The surface of OMP is mostly convex and lacking obvious small molecule binding sites, suggesting that it is more likely to be involved in modulating protein-protein interaction than in interacting with small molecule ligands. Three highly conserved regions have been identified as leading candidates for protein-protein interaction sites in OMP. One of these sites represents a loop known to mediate ligand interactions in the structurally homologous EphB2 receptor ligand-binding domain. This site is partially buried in the crystal structure but fully exposed in the NMR solution structure of OMP due to a change in the orientation of an alpha-helix that projects outward from the structurally invariant beta-clamshell core. Gating of this conformational change by molecular interactions in the signal-transduction cascade could be used to control access to OMP's equivalent of the EphB2 ligand-interaction loop, thereby allowing OMP to function as a molecular switch.