A selective membrane-targeting repurposed antibiotic with activity against persistent methicillin-resistant Staphylococcus aureus.

Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.

Hijacking the Bacterial Circuitry of Biofilm Processes via Chemical "Hot-Wiring": An Under-explored Avenue for Therapeutic Development.

Biofilm-associated infections are linked to chronic and recurring illnesses. These infections are often not susceptible to current antibiotic treatments because of the protective exocellular matrix and subpopulations of dormant or "persister" cells. Targeting bacterial circuitry involved in biofilm formation, including two-component systems, quorum sensing, polysaccharide structural integrity, and cyclic nucleotide signaling pathways, has the potential to expand the existing arsenal of therapeutics, thus catalyzing a second golden age of antibiotic development.

Polyketide synthase and non-ribosomal peptide synthetase thioesterase selectivity: logic gate or a victim of fate?

Type 1, α/ß hydrolase-like thioesterase (TE) domains are essential offloading enzymes, releasing covalently bound products from fatty acid, polyketide, and non-ribosomal peptide biosynthetic complexes. The release step can occur by attack of an exogenous nucleophile effecting hydrolysis or transesterification or by an intramolecular O-, N-, or C-nucleophile, effecting macrolactonization, macrolactamization or Claisen-like condensation of the product. Thus in addition to ensuring turnover of the pathway, TEs provide access to increased chemical diversity. We review the diversity, structure, and mechanism of PKS and NRPS TEs and discuss recent works that highlight the role of TEs as potential arbitrators in offloading. In particular, we examine cases where TEs act as logic gates that ask a particular question about the substrate and use this information to determine the substrate's fate. As the TE mechanism occurs via two steps, we analyze both the loading and release steps independently as logic gates. The use of logic gates provides an important perspective when evaluating the evolution of TEs within a pathway, as well as highlighting work towards the goal of predicting TE function in unknown and engineered pathways.

Diastereoseletive Transannular Oxa-Conjugate Addition Generates the 2,6-cis-Disubstituted Tetrahydropyran of Neopeltolide.

Transannular 2,6-disubstituted pyrans, like the one found in the cytotoxic marine natural product neopeltolide, are a key functional group in many polyketides. While oxa-conjugate additions have been shown to provide direct and rapid access to tetrahydropyrans in acyclic neopeltolide intermediates, a transannular strategy for construction of this ring system in a macrocyclic core has not been investigated. In this study, we demonstrate that a transannular oxa-conjugate addition strategy is a viable approach to the construction of the bicyclic core of neopeltolide. We show that transannular addition occurs readily with an α,ß-unsaturated ketone as the Michael acceptor and does not occur when an α,ß-unsaturated ester is the Michael acceptor. Our data indicates that oxa-conjugate addition is reversible and that the stereochemical outcome can be under thermodynamic control. Using computational chemistry, we show that the lowest energy diastereomer is the desired cis-pyran found in neopeltolide, and we experimentally demonstrate that the trans and cis diastereomers are interconvertible under reaction conditions with the cis-pyran product predominating. This oxa-conjugate addition strategy should provide a viable route to accessing the fully elaborated macrocyclic core of neopeltolide.

An evolutionary model encompassing substrate specificity and reactivity of type I polyketide synthase thioesterases.

Bacterial polyketides are a rich source of chemical diversity and pharmaceutical agents. Understanding the biochemical basis for their biosynthesis and the evolutionary driving force leading to this diversity is essential to take advantage of the enzymes as biocatalysts and to access new chemical diversity for drug discovery. Biochemical characterization of the thioesterase (TE) responsible for 6-deoxyerythronolide macrocyclization shows that a small, evolutionarily accessible change to the substrate can increase the chemical diversity of products, including macrodiolide formation. We propose an evolutionary model in which TEs are by nature non-selective for the type of chemistry they catalyze, producing a range of metabolites. As one metabolite becomes essential for improving fitness in a particular environment, the TE evolves to enrich for that corresponding reactivity. This hypothesis is supported by our phylogenetic analysis, showing convergent evolution of macrodiolide-forming TEs.

Baseline bone mineral density and boneturnover in pre-operative hip and knee arthroplasty patients.

AIMS: Osteoporosis and abnormal bone metabolism may prove to be significant factors influencing the outcome of arthroplasty surgery, predisposing to complications of aseptic loosening and peri-prosthetic fracture. We aimed to investigate baseline bone mineral density (BMD) and bone turnover in patients about to undergo arthroplasty of the hip and knee. METHODS: We prospectively measured bone mineral density of the hip and lumbar spine using dual-energy X-ray absorptiometry (DEXA) scans in a cohort of 194 patients awaiting hip or knee arthroplasty. We also assessed bone turnover using urinary deoxypyridinoline (DPD), a type I collagen crosslink, normalised to creatinine. RESULTS: The prevalence of DEXA proven hip osteoporosis (T-score ≤ -2.5) among hip and knee arthroplasty patients was found to be low at 2.8% (4 of 143). Spinal osteoporosis prevalence was higher at 6.9% (12 of 175). Sixty patients (42% (60 of 143)) had osteopenia or osteoporosis of either the hip or spine. The mean T-score for the hip was -0.34 (sd 1.23), which is within normal limits, and the mean hip Z-score was positive at 0.87 (sd 1.17), signifying higher-than-average BMD for age. The median urinary DPD/creatinine was raised in both female patients at 8.1 (interquartile range (IQR) 6.6 to 9.9) and male patients at 6.2 (IQR 4.8 to 7.5). CONCLUSIONS: Our results indicate hip and knee arthroplasty patients have higher BMD of the hip and spine compared with an age-matched general population, and a lower prevalence of osteoporosis. However, untreated osteoporotic patients are undergoing arthroplasty, which may negatively impact their outcome. Raised DPD levels suggest abnormal bone turnover, requiring further investigation. Cite this article: Bone Joint Res 2014;3:14-19.

The role of transcription in heterologous expression of polyketides in bacterial hosts.

Heterologous expression of biosynthetic pathways is an indispensable tool in the discovery, production, engineering, and characterization of bacterial polyketides and the complex enzymology involved in their biosynthesis. Ensuring transcription of polyketide biosynthetic gene clusters in heterologous hosts is a pressing problem. This review evaluates the two strategies used to ensure transcription. The first is a promoter replacement approach where promoters known to function in the heterologous host are inserted into the biosynthetic gene cluster. The second is an approach that relies on the heterologous host recognizing and utilizing promoters native to the gene cluster. Both have been successful methodologies and have different strengths and weaknesses, which are highlighted and discussed.

Selective reduction in trabecular volumetric bone mineral density during treatment for childhood acute lymphoblastic leukemia.

During treatment of childhood acute lymphoblastic leukemia (ALL) fracture incidence is increased. Studies using DXA, which measures a composite of both trabecular and cortical BMD, have shown reduced BMD during treatment. We investigated changes in compartmental (cortical and trabecular) volumetric BMD (vBMD) and bone geometry using peripheral quantitative computed tomography. These outcomes were also analysed in relation to adiposity and treatment factors. Thirty nine patients with ALL (64% male, median age 7.2 years (4.1-16.9)) were compared to 34 healthy controls (50% male, median age 9.1 years (4.4-18.7)). DXA-derived age-specific standard deviation scores (SDS) of the lumbar spine (LS) and femoral neck (FN) were reduced in subjects with ALL compared to controls (p ≤ 0.01). This persisted following adjustment for body size using height-specific SDS (LS -0.72 ± 1.02 vs -0.18 ± 0.72, p=0.01; FN -1.53 ± 0.96 vs -0.74 ± 0.74, p=0.001) and bone mineral apparent density (BMAD) SDS (LS -0.76 ± 1.14 vs 0.04 ± 1.08, p=0.01; FN -1.63 ± 1.38 vs -0.16 ± 1.20, p<0.001). Radial and tibial trabecular vBMD was also reduced (196.5 ± 54.9 mg/cm(3) vs 215.2 ± 39.9 mg/cm(3), p=0.03 and 232.8 ± 60.3mg/cm(3) vs 267.5 ± 60.2mg/cm(3), p=0.002, respectively), but cortical vBMD at the radius and tibia was similar in patients and controls. A lowered tibial bone strength index (BSI) was identified in patients with ALL (53.9 ± 23.1mg/mm(4) vs 82.5 ± 27.8 mg/mm(4), p<0.001) suggesting lower fracture threshold from compressive forces. No relationships with measures of adiposity, duration of treatment or cumulative corticosteroid dose were identified. Our findings therefore suggest that reduction in trabecular vBMD during childhood ALL treatment may contribute to the observed increased fracture incidence and bony morbidity in this group.

Clinical working postures of bachelor of oral health students.

OBJECTIVE: To observe and describe the clinical working postures of final-year Bachelor of Oral Health (BOH) students. DESIGN: Pilot observational study. SETTING: The University of Otago Faculty of Dentistry and School of Physiotherapy. METHODS: Eight final-year BOH students voluntarily participated in this study, where postural data were collected using a digital video camera during a standard clinical treatment session. The postural data were analysed using 3D Match biomechanical software. RESULTS: Final-year BOH students who work in the seated position are exposed to neck flexion of greater than 35 degrees, together with trunk flexion greater than 20 degrees and bilateral elbow flexion greater than 90 degrees. CONCLUSIONS: The findings of this study agree with the findings of previous postural studies of dental professionals. Dental hygiene students, together with their clinical supervisors, need to be aware of the importance of good working posture early in their careers, and pay particular attention to the degree of neck flexion occurring for prolonged periods.

Elastic relaxations associated with the Pm3m-R3c transition in LaAlO(3): III. Superattenuation of acoustic resonances.

Resonant ultrasound spectroscopy has been used to characterize elastic softening and anelastic dissipation processes associated with the Pm3m <--> R3c transition in single crystal and ceramic samples of LaAlO(3). Softening of the cubic structure ahead of the transition point is not accompanied by an increase in dissipation but follows different temperature dependences for the bulk modulus, (1/3)(C(11) + C(12)), and the shear components, (1/2)(C(11) + C(12)) and C(44), as if the tilting instability contains two slightly different critical temperatures. The transition itself is marked by the complete disappearance of resonance peaks (superattenuation), which then reappear below ∼700 K in spectra from single crystals. Comparisons with low frequency, high stress data from the literature indicate that the dissipation is not due to macroscopic displacement of needle twins. An alternative mechanism, local bowing of twin walls under low dynamic stress, is postulated. Pinning of the walls with respect to this displacement process occurs below ∼350 K. Anelasticity maps, analogous to plastic deformation mechanism maps, are proposed to display dispersion relations and temperature/frequency/stress fields for different twin wall related dissipation mechanisms. These allow comparisons to be made of anelastic loss mechanisms under mechanical stress with elastic behaviour observed by means of Brillouin scattering at high frequencies which might also be related to microstructure.

Elastic relaxations associated with the Pm3m-R3c transition in LaAlO(3): IV. An incipient instability below room temperature.

Resonant ultrasound spectroscopy has been used to characterize elastic softening and acoustic dissipation behaviour in single crystal and ceramic samples of LaAlO(3) between 10 and 300 K. For the twinned R3c single crystals, average values of the cubic elastic moduli (1/2)(C(11) - C(12)) and C(44) were followed while the ceramic sample provided data for the bulk and shear moduli. A Debye-like dissipation peak occurs in the vicinity of 250 K, from which an activation energy of 43 ± 6 kJ mol(-1) has been obtained. The mechanism for this is not known, but it is associated with C(44) and therefore could be related in some way to the cubic <--> rhombohedral transition at ∼817 K. Slight softening in the temperature interval ~220 --> 70 K of resonance peaks determined by shear elastic moduli hints at an incipient E(g) ferroelastic instability in LaAlO(3). The softening interval ends with a further dissipation peak at ∼60 K, the origin of which is discussed in terms of freezing of atomic motions of La and/or Al away from their high symmetry positions in the R3c structure. LaAlO(3) thus shows evidence of incipient structural instability at low temperatures which is potentially analogous with the phenomenologically rich behaviour of SrTiO(3).

Mars water-ice clouds and precipitation.

The light detection and ranging instrument on the Phoenix mission observed water-ice clouds in the atmosphere of Mars that were similar to cirrus clouds on Earth. Fall streaks in the cloud structure traced the precipitation of ice crystals toward the ground. Measurements of atmospheric dust indicated that the planetary boundary layer (PBL) on Mars was well mixed, up to heights of around 4 kilometers, by the summer daytime turbulence and convection. The water-ice clouds were detected at the top of the PBL and near the ground each night in late summer after the air temperature started decreasing. The interpretation is that water vapor mixed upward by daytime turbulence and convection forms ice crystal clouds at night that precipitate back toward the surface.

Dielectric, calorimetric and elastic anomalies associated with the first order [Formula: see text] phase transition in (Ca, Sr)TiO(3) perovskites.

Conduction calorimetry has been used to determine with high precision the latent heat and variation in heat capacity which accompany the first order [Formula: see text] phase transition in perovskites with compositions (Ca(1-x)Sr(x))TiO(3), x = 0.65, 0.68, 0.74 (CST65, CST68, CST74). In CST65 (CST68), the latent heat is dissipated/absorbed over a temperature interval of ∼11 K (∼6 K), which is centred on ∼292 K (∼258 K) during cooling and ∼302 K (∼270 K) during heating. The magnitude of the latent heat diminishes with increasing SrTiO(3) content and was not detected in CST74. Integration of the latent heat and excess heat capacity yields small excess entropies, which are consistent with the structural changes being displacive rather than order-disorder in origin. Resonant ultrasound spectroscopy measurements on the same CST65 sample as used for dielectric and calorimetric measurements through the same temperature intervals have allowed quantitative correlations to be made with the bulk modulus, shear modulus and acoustic dissipation parameter, Q(-1). The dielectric anomaly and changes in Q(-1) can be understood as being linear combinations of the properties of the separate I4/mcm and Pbcm phases in proportion to their volume fractions across the two-phase field. A change of only ∼0.5-1 GPa has been detected in the bulk modulus but the shear modulus softens by ∼5-8 GPa as the transition interval is approached from above and below. This shear mode softening presumably reflects clustering and/or phonon softening in both the I4/mcm and Pbcm structures. This pattern of structure-property relations could be typical of first order transitions in perovskites where there is no group/subgroup relationship between the high and low symmetry phases.

The extreme Kuiper Belt binary 2001 QW322.

The study of binary Kuiper Belt objects helps to probe the dynamic conditions present during planet formation in the solar system. We report on the mutual-orbit determination of 2001 QW322, a Kuiper Belt binary with a very large separation whose properties challenge binary-formation and -evolution theories. Six years of tracking indicate that the binary's mutual-orbit period is approximately 25 to 30 years, that the orbit pole is retrograde and inclined 50 degrees to 62 degrees from the ecliptic plane, and, most surprisingly, that the mutual orbital eccentricity is <0.4. The semimajor axis of 105,000 to 135,000 kilometers is 10 times that of other near-equal-mass binaries. Because this weakly bound binary is prone to orbital disruption by interlopers, its lifetime in its present state is probably less than 1 billion years.

Detection of stratospheric ozone intrusions by windprofiler radars.

Stratospheric ozone attenuates harmful ultraviolet radiation and protects the Earth's biosphere. Ozone is also of fundamental importance for the chemistry of the lowermost part of the atmosphere, the troposphere. At ground level, ozone is an important by-product of anthropogenic pollution, damaging forests and crops, and negatively affecting human health. Ozone is critical to the chemical and thermal balance of the troposphere because, via the formation of hydroxyl radicals, it controls the capacity of tropospheric air to oxidize and remove other pollutants. Moreover, ozone is an important greenhouse gas, particularly in the upper troposphere. Although photochemistry in the lower troposphere is the major source of tropospheric ozone, the stratosphere-troposphere transport of ozone is important to the overall climatology, budget and long-term trends of tropospheric ozone. Stratospheric intrusion events, however, are still poorly understood. Here we introduce the use of modern windprofiler radars to assist in such transport investigations. By hourly monitoring the radar-derived tropopause height in combination with a series of frequent ozonesonde balloon launches, we find numerous intrusions of ozone from the stratosphere into the troposphere in southeastern Canada. On some occasions, ozone is dispersed at altitudes of two to four kilometres, but on other occasions it reaches the ground, where it can dominate the ozone density variability. We observe rapid changes in radar tropopause height immediately preceding these intrusion events. Such changes therefore serve as a valuable diagnostic for the occurrence of ozone intrusion events. Our studies emphasize the impact that stratospheric ozone can have on tropospheric ozone, and show that windprofiler data can be used to infer the possibility of ozone intrusions, as well as better represent tropopause motions in association with stratosphere-troposphere transport.

Migration of interplanetary dust.

We numerically investigate the migration of dust particles with initial orbits close to those of the numbered asteroids, observed trans-Neptunian objects, and comet Encke. The fraction of silicate asteroidal particles that collided with the Earth during their lifetime varied from 11% for 100 micron particles to 0.008% for 1 micron particles. Almost all asteroidal particles with diameter d >/= 4 microns collided with the Sun. For migrating asteroidal dust particles, the peaks in semimajor axis distribution at the n:(n + 1) resonances with Earth and Venus, and the gaps associated with the 1:1 resonances with these planets are more pronounced for larger particles. The probability of collisions of cometary particles with the Earth is smaller than for asteroidal particles, and this difference is greater for larger particles.

CD47 (integrin-associated protein) engagement of dendritic cell and macrophage counterreceptors is required to prevent the clearance of donor lymphohematopoietic cells.

Integrin-associated protein (CD47) is a broadly expressed protein that costimulates T cells, facilitates leukocyte migration, and inhibits macrophage scavenger function. To determine the role of CD47 in regulating alloresponses, CD47(+/+) or CD47(-/-) T cells were infused into irradiated or nonconditioned major histocompatibility complex disparate recipients. Graft-versus-host disease lethality was markedly reduced with CD47(-/-) T cells. Donor CD47(-/-) T cells failed to engraft in immunodeficient allogeneic recipients. CD47(-/-) marrow was unable to reconstitute heavily irradiated allogeneic or congenic immune-deficient CD47(+/+) recipients. These data suggested that CD47(-/-) T cells and marrow cells were cleared by the innate immune system. To address this hypothesis, dye-labeled CD47(-/-) and CD47(+/+) lymphocytes or marrow cells were infused in vivo and clearance was followed. Dye-labeled CD47(-/-) cells were engulfed by splenic dendritic cells and macrophages resulting in the clearance of virtually all CD47(-/-) lymphohematopoietic cells within 1 day after infusion. Host phagocyte-depleted CD47(+/+) recipients partially accepted allogeneic CD47(-/-) T cells. Thus, dendritic cells and macrophages clear lymphohematopoietic cells that have downregulated CD47 density. CD47 expression may be a critical indicator for determining whether lymphohematopoietic cells will survive or be cleared.

Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in naive and bone marrow transplant recipients.

Immunostimulatory cytosine-phophate-guanosine (CpG)--containing motifs in bacterial DNA are potent immune system activators. Depending on the bases flanking the CpG motif and on the DNA backbone, CpG oligodeoxynucleotides (ODNs) can induce relatively more B-cell activation or relatively more natural killer (NK)--cell activation. To evaluate their antitumor activities, an NK-optimized ODN (1585) and 2 B-cell--optimized ODNs (1826 and 2006) were compared for their ability to protect naive mice against a lethal acute myelogenous leukemia (AML) challenge. CpG 2006, but not CpG 1585, administered 2 days before the AML challenge, allowed mice to survive more than 100 times a lethal tumor dose. Cell depletion studies showed that protection did not require T or B cells but depended on NK cells and also on an NK-independent mechanism. CpG 2006 protected against AML challenge in both syngeneic and allogeneic bone marrow transplant (BMT) recipients at both early and late time points after transplantation. Although CpG 1585 had no protective effect on its own, it showed a striking synergy with CpG 2006 to induce prolonged survival to AML challenge in allogeneic recipients of T-cell-depleted marrow grafts, exceeding the survival benefit of donor lymphocyte infusion (DLI). When combined with DLI, a synergistic effect was observed in recipients of CpG2006 or 2006 + 1585 with 88% of mice surviving long-term. These data are the first to indicate that the systemic administration of CpG ODNs is a potent means of inducing therapeutic anti-AML innate immune responses in naive and BMT recipients. (Blood. 2001;98:1217-1225)

Requirements for the promotion of allogeneic engraftment by anti-CD154 (anti-CD40L) monoclonal antibody under nonmyeloablative conditions.

The promotion of alloengraftment in the absence of global immune suppression and multiorgan toxicity is a major goal of transplantation. It is demonstrated that the infusion of a single modest bone marrow dosage in 200 cGy-irradiated recipients treated with anti-CD154 (anti-CD40L) monoclonal antibody (mAb) resulted in chimerism levels of 48%. Reducing irradiation to 100 or 50 cGy permitted 24% and 10% chimerism, respectively. In contrast, pan-T-cell depletion resulted in only transient engraftment in 200 cGy-irradiated recipients. Host CD4(+) cells were essential for alloengraftment as depletion of CD4(+) cells abrogated engraftment in anti-CD154-treated recipients. Strikingly, the depletion of CD8(+) cells did not further enhance engraftment in anti-CD154 mAb-treated recipients in a model in which rejection is mediated by both CD4(+) and CD8(+) T cells. However, anti-CD154 mAb did facilitate engraftment in a model in which only CD8(+) T cells mediate rejection. Furthermore, CD154 deletional mice irradiated with 200 cGy irradiation were not tolerant of grafts, suggesting that engraftment promotion by anti-CD154 mAb may not simply be the result of CD154:CD40 blockade. Together, these data suggest that a CD4(+) regulatory T cell may be induced by anti-CD154 mAb. In contrast to anti-CD154 mAb, anti-B7 mAb did not promote donor engraftment. Additionally, the administration of either anti-CD28 mAb or anti-CD152 (anti-CTLA-4) mAb or the use of CD28 deletional recipients abrogated engraftment in anti-CD154 mAb-treated mice, suggesting that balanced CD28/CD152:B7 interactions are required for the engraftment-promoting capacity of anti-CD154 mAb. These data have important ramifications for the design of clinical nonmyeloablative regimens based on anti-CD154 mAb administration.

CD4(+)CD25(+) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade.

Immune regulatory CD4(+)CD25(+) cells play a vital role in the induction and maintenance of self-tolerance and are essential for T cell homeostasis and the prevention of autoimmunity. Induction of tolerance to allogeneic donor grafts is a clinically desirable goal in bone marrow and solid organ transplantation. To determine whether CD4(+)CD25(+) cells regulate T cell responses to alloantigen and are critical for tolerance induction, murine CD4(+) T cells were tolerized to alloantigen via ex vivo CD40 ligand (CD40L)/CD40 or CD28/cytotoxic T lymphocyte-associated antigen 4/B7 blockade resulting in secondary mixed leukocyte reaction hyporesponsiveness and tolerance to alloantigen in vivo. CD4(+)CD25(+) T cells were found to be potent regulators of alloresponses. Depletion of CD4(+)CD25(+) T cells from the CD4(+) responder population completely abrogated ex vivo tolerance induction to alloantigen as measured by intact responses to alloantigen restimulation in vitro and in vivo. Addback of CD4(+)CD25(+) T cells to CD4(+)CD25(-) cultures restored tolerance induction. These data are the first to indicate that CD4(+)CD25(+) cells are essential for the induction of tolerance to alloantigen and have important implications for tolerance-inducing strategies targeted at T cell costimulatory pathways.