Developing a Natural History Model for Duchenne Muscular Dystrophy.

BACKGROUND: The aim of this study was to pool multiple data sets to build a patient-centric, data-informed, natural history model (NHM) for Duchenne muscular dystrophy (DMD) to estimate disease trajectory across patient lifetime under current standard of care in future economic evaluations. The study was conducted as part of Project HERCULES, a multi-stakeholder collaboration to develop tools to support health technology assessments of new treatments for DMD. METHODS: Health states were informed by a review of NHMs for DMD and input from clinicians, patients and caregivers, and defined using common outcomes in clinical trials and real-world practice. The primary source informing the NHM was the Critical Path Institute Duchenne Regulatory Science Consortium (D-RSC) database. This was supplemented with expert input obtained via an elicitation exercise, and a systematic literature review and meta-analysis of mortality data. RESULTS: The NHM includes ambulatory, transfer and non-ambulatory phases, which capture loss of ambulation, ability to weight bear and upper body and respiratory function, respectively. The NHM estimates patients spend approximately 9.5 years in ambulatory states, 1.5 years in the transfer state and the remainder of their lives in non-ambulatory states. Median predicted survival is 34.8 years (95% CI 34.1-35.8). CONCLUSION: The model includes a detailed disease pathway for DMD, including the clinically and economically important transfer state. The NHM may be used to estimate the current trajectory of DMD in economic evaluations of new treatments, facilitating inclusion of a lifetime time horizon, and will help identify areas for further research.

In vitro antimicrobial activity of crude propolis extracts and fractions.

The search for antimicrobials in propolis presents a new dimension for addressing the problem of antimicrobial drug resistance. The aim of this study was to determine the antimicrobial activity of extracts of crude propolis collected from different regions in Ghana and their active fractions. The antimicrobial activity of the extracts, as well as that of the chloroform, ethyl acetate, and petroleum ether fractions of the active samples were determined using the agar well diffusion method. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the most active fractions were determined. The various crude propolis extracts frequently produced zones of inhibition against Staphylococcus aureus (17/20) than Pseudomonas aeruginosa (16/20), and Escherichia coli (1/20) test isolates. Chloroform and ethyl acetate solvents produced fractions possessing greater antimicrobial activity than the petroleum ether fraction. The mean MIC range of the most active fractions was greatest for S. aureus (76.0 ± 34.8-48.0 ± 33.0 mg/ml) than for P. aeruginosa (40.8 ± 33.3-30.4 ± 6.7 mg/ml) and E. coli, as was the mean MBC. Propolis has antimicrobial potential, and hence should be exploited as an alternative for the treatment of bacterial infections.

CD4+ T Cells Are Dispensable for Induction of Broad Heterologous HIV Neutralizing Antibodies in Rhesus Macaques.

Induction of broadly neutralizing antibodies (bNAbs) is a major goal for HIV vaccine development. HIV envelope glycoprotein (Env)-specific bNAbs isolated from HIV-infected individuals exhibit substantial somatic hypermutation and correlate with T follicular helper (Tfh) responses. Using the VC10014 DNA-protein co-immunization vaccine platform consisting of gp160 plasmids and gp140 trimeric proteins derived from an HIV-1 infected subject that developed bNAbs, we determined the characteristics of the Env-specific humoral response in vaccinated rhesus macaques in the context of CD4+ T cell depletion. Unexpectedly, both CD4+ depleted and non-depleted animals developed comparable Tier 1 and 2 heterologous HIV-1 neutralizing plasma antibody titers. There was no deficit in protection from SHIV challenge, no diminution of titers of HIV Env-specific cross-clade binding antibodies, antibody dependent cellular phagocytosis, or antibody-dependent complement deposition in the CD4+ depleted animals. These collective results suggest that in the presence of diminished CD4+ T cell help, HIV neutralizing antibodies were still generated, which may have implications for developing effective HIV vaccine strategies.

Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters.

SARS-CoV-2 infection results in viral burden in the respiratory tract, enabling transmission and leading to substantial lung pathology. The 1212C2 fully human monoclonal antibody was derived from an IgM memory B cell of a COVID-19 patient, has high affinity for the Spike protein receptor binding domain, neutralizes SARS-CoV-2, and exhibits in vivo prophylactic and therapeutic activity in hamsters when delivered intraperitoneally, reducing upper and lower respiratory viral burden and lung pathology. Inhalation of nebulized 1212C2 at levels as low as 0.6 mg/kg, corresponding to 0.03 mg/kg lung-deposited dose, reduced the viral burden below the detection limit and mitigated lung pathology. The therapeutic efficacy of an exceedingly low dose of inhaled 1212C2 supports the rationale for local lung delivery for dose-sparing benefits, as compared to the conventional parenteral route of administration. These results suggest that the clinical development of 1212C2 formulated and delivered via inhalation for the treatment of SARS-CoV-2 infection should be considered.

Mathematical Discrepancies of the Tono-Pen Applanation Tonometer.

PURPOSE OF THE STUDY: The purpose of the study was to determine if Tono-Pen tonometers use simple average and coefficient of variation (CV) algorithms to calculate intraocular pressure (IOP). MATERIAL AND METHODS: IOPs were measured as part of routine ocular examination in 152 client-owned dogs. Using 11 Tono-Pen's, a total of 778 averaged readings were collected. Individual IOP readings, and average IOP and CV displayed by the instrument, were recorded. Average IOP and CV were then manually calculated from individual readings and compared with those displayed by the instrument. RESULTS: The mean absolute difference between the calculated and displayed average IOP was 1.37±2.01 mm Hg (P<0.001). In 6% of cases, the calculated average IOP was 5 to 15 mm Hg different from the displayed average IOP. The difference between the displayed and calculated average IOP was significantly higher in hypertensive eyes with displayed IOP≥25 mm Hg. Calculated CV was equal to, lower than, or greater than displayed CV in 28.6%, 1.5%, and 69.7% of cases, respectively. In 17.6% of cases, calculated CV was >20%, but displayed CV was <5%. Receiver operating characteristic analysis could not correlate number of individual IOP readings with magnitude of difference in average IOP. CONCLUSIONS: Calculated average IOP and CV differ significantly from values displayed by the instrument, especially at higher IOPs. A difference of ≥5 mm Hg between calculated and displayed average IOP seen in 6% of cases may impact clinical judgement. Displayed CV<5% does not correlate with accurate IOP measurement based on individual results.

Effects of intramuscular injection of glycopyrrolate on Schirmer tear test I results in dogs.

OBJECTIVE To determine effects of glycopyrrolate administered IM on Schirmer tear test I (STT I) measurements in dogs. DESIGN Prospective clinical study. ANIMALS 13 client- and staff-owned dogs. PROCEDURES For both eyes of each dog, STT I measurements were recorded twice 20 minutes apart (at T1 and T2) and 2 to 4 hours later (at T3). Glycopyrrolate (0.01 mg/kg [0.005 mg/lb]) was administered IM to all dogs (3 dogs received an injection of saline [0.9% NaCl] solution on an earlier occasion), and final STT I measurements were recorded 20 minutes later (at T4). Intraocular pressures, heart rate, and respiratory rate were also recorded at each time point. RESULTS Ophthalmic variables did not differ between right and left eyes. In all dogs, variables at T1, T2, or T3 (measurements before glycopyrrolate administration) did not differ; baseline values were therefore defined at T3. At T4, STT I measurements were significantly decreased (mean ± SD decrease, 67.4 ± 15.4% [mean actual decrease, 15.8 mm/min]). During the same period, mean heart rate increased by 26.5 ± 12.0% (mean actual increase, 30.2 beats/min). Glycopyrrolate had no effect on intraocular pressure or respiratory rate. In 5 dogs at 24 hours after glycopyrrolate treatment, STT I measurement in each eye had returned to baseline value. Saline solution treatment (3 dogs) had no effect on any variables. CONCLUSIONS AND CLINICAL RELEVANCE In dogs, IM injection of glycopyrrolate resulted in a clinically relevant transient decrease in aqueous tear production. Application of lacrimomimetics beginning at the time of or within 20 minutes after glycopyrrolate premedication is recommended until STT I measurements return to baseline.

Laboratory-based nationwide surveillance of antimicrobial resistance in Ghana.

Global efforts are underway to combat antimicrobial resistance (AMR). A key target in this intervention is surveillance for local and national action. Data on AMR in Ghana are limited, and monitoring of AMR is nonexistent. We sought to generate baseline data on AMR, and to assess the readiness of Ghana in laboratory-based surveillance. Biomedical scientists in laboratories across Ghana with capacity to perform bacteriological culture were selected and trained. In-house standard operating protocols were used to perform microbiological investigations on clinical specimens. Additional microbiological tests and data analyses were performed at a centralized laboratory. Surveillance data were stored and analyzed using WHONET program files. A total of 24 laboratories participated in the training, and 1,598 data sets were included in the final analysis. A majority of the bacterial species were isolated from outpatients (963 isolates; 60.3%). Urine (617 isolates; 38.6%) was the most common clinical specimen cultured, compared to blood (100 isolates; 6.3%). Ten of 18 laboratories performed blood culture. Bacteria isolated included Escherichia coli (27.5%), Pseudomonas spp. (14.0%), Staphylococcus aureus (11.5%), Streptococcus spp. (2.3%), and Salmonella enterica serovar Typhi (0.6%). Most of the isolates were multidrug-resistant, and over 80% of them were extended-spectrum beta-lactamases-producing. Minimum inhibitory concentration levels at 50% and at 90% for ciprofloxacin, ceftriaxone, and amikacin on selected multidrug-resistant bacteria species ranged between 2 µg/mL and >256 µg/mL. A range of clinical bacterial isolates were resistant to important commonly used antimicrobials in the country, necessitating an effective surveillance to continuously monitor AMR in Ghana. With local and international support, Ghana can participate in global AMR surveillance.

An allolactose trapped at the lacZ ß-galactosidase active site with its galactosyl moiety in a (4)H3 conformation provides insights into the formation, conformation, and stabilization of the transition state.

When lactose was incubated with G794A-ß-galactosidase (a variant with a "closed" active site loop that binds transition state analogs well) an allolactose was trapped with its Gal moiety in a (4)H3 conformation, similar to the oxocarbenium ion-like conformation expected of the transition state. The numerous interactions formed between the (4)H3 structure and ß-galactosidase indicate that this structure is representative of the transition state. This conformation is also very similar to that of d-galactono-1,5-lactone, a good transition state analog. Evidence indicates that substrates take up the (4)H3 conformation during migration from the shallow to the deep mode. Steric forces utilizing His418 and other residues are important for positioning the O1 leaving group into a quasi-axial position. An electrostatic interaction between the O5 of the distorted Gal and Tyr503 as well as C-H-π bonds with Trp568 are also significant. Computational studies of the energy of sugar ring distortion show that the ß-galactosidase reaction itinerary is driven by energetic considerations in utilization of a (4)H3 transition state with a novel (4)C1-(4)H3-(4)C1 conformation itinerary. To our knowledge, this is the first X-ray crystallographic structural demonstration that the transition state of a natural substrate of a glycosidase has a (4)H3 conformation.

Multidrug-Resistant Streptococcus pneumoniae Isolates from Healthy Ghanaian Preschool Children.

Streptococcus pneumoniae is the cause of high mortality among children worldwide. Antimicrobial treatment and vaccination are used to control pneumococcal infections. In Ghana, data on antimicrobial resistance and the prevalence of multidrug-resistant pneumococcal clones are scarce; hence, the aim of this study was to determine the antibiogram of S. pneumoniae recovered from Ghanaian children younger than six years of age and to what extent resistances were due to the spread of certain sero- and multilocus sequence typing (MLST) types. The susceptibility of 115 pneumococcal isolates, recovered in a previous study, to six antimicrobials was determined by disk diffusion test. Overall, 90.4% of isolates were intermediate penicillin resistant, 99.1% were trimethoprim resistant, 73.0% were tetracycline resistant, and 33.9% were sulfamethoxazole resistant. Low resistance was recorded for erythromycin (2.6%) and cefotaxime (5.2%). Overall, 72.2% of isolates were resistant to penicillin (I or R) and at least two other antimicrobials. MLST of 20 isolates showing resistance to at least four antimicrobials revealed a high diversity documented by 16 different clones, none of which had previously been associated with multidrug resistance. The resistances found may have emerged due to nonprudent antimicrobial use practices and there is a need to monitor and promote prudent antimicrobial usage in Ghana.

Elucidating factors important for monovalent cation selectivity in enzymes: E. coli ß-galactosidase as a model.

Many enzymes require a specific monovalent cation (M(+)), that is either Na(+) or K(+), for optimal activity. While high selectivity M(+) sites in transport proteins have been extensively studied, enzyme M(+) binding sites generally have lower selectivity and are less characterized. Here we study the M(+) binding site of the model enzyme E. coli ß-galactosidase, which is about 10 fold selective for Na(+) over K(+). Combining data from X-ray crystallography and computational models, we find the electrostatic environment predominates in defining the Na(+) selectivity. In this lower selectivity site rather subtle influences on the electrostatic environment become significant, including the induced polarization effects of the M(+) on the coordinating ligands and the effect of second coordination shell residues on the charge distribution of the primary ligands. This work expands the knowledge of ion selectivity in proteins to denote novel mechanisms important for the selectivity of M(+) sites in enzymes.

Penicillin resistance and serotype distribution of Streptococcus pneumoniae in Ghanaian children less than six years of age.

BACKGROUND: The objective of this study was to determine the prevalence of nasopharyngeal carriage, serotype distribution, and penicillin resistance of Streptococcus pneumoniae in children ≤ 6 years of age in Ghana. METHODS: A cross-sectional study was carried out on a cluster-randomized sample of children ≤ 6 years of age attending nurseries and kindergartens in Accra and Tamale, Ghana. Basic data on age, sex and exposure to antimicrobials in the previous month were collected on all study subjects. Nasopharyngeal swabs were obtained from participants and all pneumococcal isolates were characterized by serotyping and their penicillin resistance determined. RESULTS: The overall prevalence of pneumococcal carriage among the children was 34% in Accra and 31% in Tamale. The predominant serotypes were 19F, 6B, 23F, and 6A with 23% of the isolates being non-typable in Accra and 12% in Tamale. Only two isolates (serotypes 19F and 6B) from Tamale had a MIC >2 µg/ml and were classified as fully penicillin resistant with 45% of the isolates having intermediate resistance. CONCLUSIONS: These findings indicate that the 13-valent pneumococcal conjugate vaccine (PCV-13) recently introduced in Ghana will cover 48% and 51% of the serotypes identified in Accra and Tamale, respectively. The 23-valent pneumococcal polysaccharide vaccine (PPV-23) will cover 54% of all serotypes detected. The two penicillin resistant isolates (MIC 32 µg/ml) were serotypes included in both PCV-13 and PPV-23. A nationwide monitoring system of penicillin susceptibility patterns and pneumococcal serotypes is recommended.

Biochemical and biophysical properties of a putative hub protein expressed by vaccinia virus.

H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.

Effects of anesthetic induction with midazolam-propofol and midazolam-etomidate on selected ocular and cardiorespiratory variables in clinically normal dogs.

OBJECTIVE: To compare effects of anesthetic induction with midazolam-propofol or midazolam-etomidate on intraocular pressure (IOP), pupillary diameter (PD), pulse rate, blood pressure, and respiratory rate in clinically normal dogs. ANIMALS: 18 dogs. PROCEDURES: Dogs undergoing ophthalmic surgery received midazolam (0.2 mg/kg, IV) and either propofol or etomidate (IV) until intubatable. For all dogs, results of physical examinations, ophthalmic examinations of the nonoperated eye, and preanesthetic blood analyses were normal. Intraocular pressure, PD, blood pressure, pulse rate, and respiratory rate were measured in the nonoperated eye at 5 time points: just prior to the anesthetic induction sequence, after 5 minutes of preanesthetic oxygenation via face mask, after IV administration of midazolam, after IV anesthetic induction, and after endotracheal intubation. RESULTS: PD decreased significantly from baseline by 4.4 ± 0.4 mm (mean ± SD) after anesthetic induction and 5.3 ± 0.4 mm after intubation in the etomidate group and by 1. 2 ± 0.4 mm after intubation in the propofol group. Intraocular pressure was increased significantly from baseline by 3.2 ± 1.0 mm Hg after anesthetic induction in the etomidate group and by 4.7 ± 1.2 mm Hg after anesthetic induction and 4.5 ± 1. 2 mm Hg after intubation in the propofol group. Pulse rate was significantly lower by 28.6 ± 12.6 beats/min after anesthetic induction in the etomidate group, compared with the propofol group. CONCLUSIONS AND CLINICAL RELEVANCE: At the studied doses, midazolam-etomidate caused clinically important miosis and increased IOP. Midazolam-propofol caused an even greater increase in IOP but had minimal effects on PD.

Structural explanation for allolactose (lac operon inducer) synthesis by lacZ ß-galactosidase and the evolutionary relationship between allolactose synthesis and the lac repressor.

ß-Galactosidase (lacZ) has bifunctional activity. It hydrolyzes lactose to galactose and glucose and catalyzes the intramolecular isomerization of lactose to allolactose, the lac operon inducer. ß-Galactosidase promotes the isomerization by means of an acceptor site that binds glucose after its cleavage from lactose and thus delays its exit from the site. However, because of its relatively low affinity for glucose, details of this site have remained elusive. We present structural data mapping the glucose site based on a substituted enzyme (G794A-ß-galactosidase) that traps allolactose. Various lines of evidence indicate that the glucose of the trapped allolactose is in the acceptor position. The evidence includes structures with Bis-Tris (2,2-bis(hydroxymethyl)-2,2',2″-nitrilotriethanol) and L-ribose in the site and kinetic binding studies with substituted ß-galactosidases. The site is composed of Asn-102, His-418, Lys-517, Ser-796, Glu-797, and Trp-999. Ser-796 and Glu-797 are part of a loop (residues 795-803) that closes over the active site. This loop appears essential for the bifunctional nature of the enzyme because it helps form the glucose binding site. In addition, because the loop is mobile, glucose binding is transient, allowing the release of some glucose. Bioinformatics studies showed that the residues important for interacting with glucose are only conserved in a subset of related enzymes. Thus, intramolecular isomerization is not a universal feature of ß-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of ß-galactosidase played an important role in lac operon evolution.

LacZ ß-galactosidase: structure and function of an enzyme of historical and molecular biological importance.

This review provides an overview of the structure, function, and catalytic mechanism of lacZ ß-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α-complementation, in which addition to the inactive dimers of peptides containing the "missing" N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of ß-galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon.

Substitution for Asn460 cripples ß-galactosidase (Escherichia coli) by increasing substrate affinity and decreasing transition state stability.

Substrate initially binds to ß-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ∼3Å to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site. Structural and kinetic studies were done with ß-galactosidases with substitutions for Asn460. The substituted enzymes have enhanced substrate affinity in the shallow site indicating lower E·substrate complex energy levels. They have poor transition state stabilization in the deep site that is manifested by increased energy levels of the E·transition state complexes. These changes in stability result in increased activation energies and lower k(cat) values. Substrate affinity to N460D-ß-galactosidase was enhanced through greater binding enthalpy (stronger H-bonds through the bridging water) while better affinity to N460T-ß-galactosidase occurred because of greater binding entropy. The transition states are less stable with N460S- and N460T-ß-galactosidase because of the weakening or loss of the important bond to the O2 hydroxyl of the transition state. For N460D-ß-galactosidase, the transition state is less stable due to an increased entropy penalty.

Ser-796 of ß-galactosidase (Escherichia coli) plays a key role in maintaining a balance between the opened and closed conformations of the catalytically important active site loop.

A loop (residues 794-803) at the active site of ß-galactosidase (Escherichia coli) opens and closes during catalysis. The α and ß carbons of Ser-796 form a hydrophobic connection to Phe-601 when the loop is closed while a connection via two H-bonds with the Ser hydroxyl occurs with the loop open. ß-Galactosidases with substitutions for Ser-796 were investigated. Replacement by Ala strongly stabilizes the closed conformation because of greater hydrophobicity and loss of H-bonding ability while replacement with Thr stabilizes the open form through hydrophobic interactions with its methyl group. Upon substitution with Asp much of the defined loop structure is lost. The different open-closed equilibria cause differences in the stabilities of the enzyme·substrate and enzyme·transition state complexes and of the covalent intermediate that affect the activation thermodynamics. With Ala, large changes of both the galactosylation (k(2)) and degalactosylation (k(3)) rates occur. With Thr and Asp, the k(2) and k(3) were not changed as much but large ΔH(‡) and TΔS(‡) changes showed that the substitutions caused mechanistic changes. Overall, the hydrophobic and H-bonding properties of Ser-796 result in interactions strong enough to stabilize the open or closed conformations of the loop but weak enough to allow loop movement during the reaction.

Importance of Arg-599 of ß-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop.

Structural and kinetic data show that Arg-599 of ß-galactosidase plays an important role in anchoring the "open" conformations of both Phe-601 and an active-site loop (residues 794-803). When alanine was substituted for Arg-599, the conformations of Phe-601 and the loop shifted towards the "closed" positions because interactions with the guanidinium side chain were lost. Also, Phe-601, the loop, and Na+, which is ligated by the backbone carbonyl of Phe-601, lost structural order, as indicated by large B-factors. IPTG, a substrate analog, restored the conformations of Phe-601 and the loop of R599A-ß-galactosidase to the open state found with IPTG-complexed native enzyme and partially reinstated order. ᴅ-Galactonolactone, a transition state analog, restored the closed conformations of R599A-ß-galactosidase to those found with ᴅ-galactonolactone-complexed native enzyme and completely re-established the order. Substrates and substrate analogs bound R599A-ß-galactosidase with less affinity because the closed conformation does not allow substrate binding and extra energy is required for Phe-601 and the loop to open. In contrast, transition state analog binding, which occurs best when the loop is closed, was several-fold better. The higher energy level of the enzyme•substrate complex and the lower energy level of the first transition state means that less activation energy is needed to form the first transition state and thus the rate of the first catalytic step (k2) increased substantially. The rate of the second catalytic step (k3) decreased, likely because the covalent form is more stabilized than the second transition state when Phe-601 and the loop are closed. The importance of the guanidinium group of Arg-599 was confirmed by restoration of conformation, order, and activity by guanidinium ions.

Role of Met-542 as a guide for the conformational changes of Phe-601 that occur during the reaction of β-galactosidase (Escherichia coli).

The Met-542 residue of ß-galactosidase is important for the enzyme's activity because it acts as a guide for the movement of the benzyl side chain of Phe-601 between two stable positions. This movement occurs in concert with an important conformational change (open vs. closed) of an active site loop (residues 794-803). Phe-601 and Arg-599, which interact with each other via the π electrons of Phe-601 and the guanidium cation of Arg-599, move out of their normal positions and become disordered when Met-542 is replaced by an Ala residue because of the loss of the guide. Since the backbone carbonyl of Phe-601 is a ligand for Na(+), the Na(+) also moves out of its normal position and becomes disordered; the Na(+) binds about 120 times more poorly. In turn, two other Na(+) ligands, Asn-604 and Asp-201, become disordered. A substrate analog (IPTG) restored Arg-599, Phe-601, and Na(+) to their normal open-loop positions, whereas a transition state analog d-galactonolactone) restored them to their normal closed-loop positions. These compounds also restored order to Phe-601, Asn-604, Asp-201, and Na(+). Binding energy was, however, necessary to restore structure and order. The K(s) values of oNPG and pNPG and the competitive K(i) values of substrate analogs were 90-250 times higher than with native enzyme, whereas the competitive K(i) values of transition state analogs were ~3.5-10 times higher. Because of this, the E•S energy level is raised more than the E•transition state energy level and less activation energy is needed for galactosylation. The galactosylation rates (k2) of M542A-ß-galactosidase therefore increase. However, the rate of degalactosylation (k3) decreased because the E•transition state complex is less stable.

Imaging of peritoneal catheter tunnel infection using positron-emission tomography.

Imaging by ultrasonography or scintigraphy may assist in the diagnosis and management of tunnel infections of the peritoneal dialysis (PD) catheter. Here, we report a case of tunnel infection in which imaging with positron-emission tomography (PET) correctly predicted failure of conservative management. A 61-year-old man with diabetic nephropathy commenced PD in January 2008. He developed erythema and drainage at the exit site, with negative cultures in February 2008, and frank exit-site infection (ESI) with purulent drainage growing methicillin-sensitive Staphylococcus aureus [MSSA (treated with 3 weeks of oral dicloxacillin)] in August 2008. Subsequently, MSSA-growing purulent drainage from the exit site persisted. Systemic antibiotics were not administered, but there was gradual improvement with gentamicin ointment alone. In November 2008, the patient developed partial extrusion of the outer cuff of the PD catheter. In January 2009, a new ESI developed. Despite a week of treatment with cefazolin and gentamicin, the patient still developed his first episode of peritonitis with coagulase-negative Staphylococcus. He then received intraperitoneal vancomycin with good response. Although the ESI appeared to have responded to the treatment, PET imaging showed increased fludeoxyglucose (FDG) activity in the whole abdominal wall portion of the PD catheter. The patient resisted removal of the catheter and had no further signs of infection until June 2009. At that time he presented with exuberant inflammatory tissue ("proud flesh") at the exit site. Repeated PET imaging again showed increased FDG activity along the abdominal wall portion of the catheter. The PD catheter was removed and found to be infected. The patient was placed on temporary hemodialysis. This case demonstrates that PET imaging, in addition to other imaging techniques, may be useful for diagnosing and managing PD catheter infections.