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SARS-CoV-2 transmission is largely driven by heterogeneous dynamics at a local scale, leaving local health departments to design interventions with limited information. We analyzed SARS-CoV-2 genomes sampled between February 2020 and March 2022 jointly with epidemiological and cell phone mobility data to investigate fine scale spatiotemporal SARS-CoV-2 transmission dynamics in King County, Washington, a diverse, metropolitan US county. We applied an approximate structured coalescent approach to model transmission within and between North King County and South King County alongside the rate of outside introductions into the county. Our phylodynamic analyses reveal that following stay-at-home orders, the epidemic trajectories of North and South King County began to diverge. We find that South King County consistently had more reported and estimated cases, COVID-19 hospitalizations, and longer persistence of local viral transmission when compared to North King County, where viral importations from outside drove a larger proportion of new cases. Using mobility and demographic data, we also find that South King County experienced a more modest and less sustained reduction in mobility following stay-at-home orders than North King County, while also bearing more socioeconomic inequities that might contribute to a disproportionate burden of SARS-CoV-2 transmission. Overall, our findings suggest a role for local-scale phylodynamics in understanding the heterogeneous transmission landscape.One Sentence SummaryAnalysis of SARS-CoV-2 genomes in King County, Washington show that diverse areas in the same metropolitan region can have different epidemic dynamics.

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against SARS-CoV-2 and HIV-1. Yet, its primary receptor portfolio remains controversial, potentially including sialic acid, CAR, integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned by the inability to co-crystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infectionin vitroandin vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domains CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (IM) injection, but not after intranasal (IN) administration. Ad26 transduction was 10-fold lower than Ad5 after intratumoral (IT) injection of CD46-expressing tumors. Ad26 transduction of liver was 1000-fold lower than Ad5 after intravenous (IV) injection. These data demonstrate the use of CD46 by Ad26 under certain situations, but also show that the receptor has little consequence by other routes of administration. Finally, IV injection of high doses of Ad26 into CD46 mice induced release of liver enzymes in the bloodstream and reduced white blood cell counts, but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during COVID-19 vaccination with this serotype of adenovirus.IMPORTANCEHuman species D Ad26 is being pursued as a low seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection, and its role in virus biology, vaccine efficacy, and importantly, in vaccine safety.

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host., Author summary Syphilis continues to be a disease of global and public health concern, even though the infection can be easily diagnosed and effectively treated with penicillin. Although infected individuals often develop a strong immunity to the pathogen, repeated infection with the syphilis agent, Treponema pallidum subspecies pallidum (T. pallidum), is possible. Several studies point at antigenic variation of the T. pallidum TprK protein as the mechanism responsible for evasion of the immunity that develops during infection, pathogen persistence, and re-infection. Past studies have highlighted the importance of immune clearance of dominant variants that, in turn, allows less represented variants to emerge. The contribution of an immunity-independent baseline generation of variability in the tprK gene is less clear. Here, we used deep sequencing to profile tprK variants using a laboratory-isolated T. pallidum strain nearly isogenic for tprK that was propagated over time in vitro, where no immune pressure is exerted on the pathogen, as well as in samples obtained from immunosuppressed and immunocompetent rabbits infected with the same strain. We confirmed that tprK accumulates significantly more diversity under immune pressure, and demonstrated a low but discernible basal rate of gene conversion in complete absence of immune pressure.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic painful skin disease that severely impairs patients’ quality of life. While high quality trials of HS therapies remain limited, medical knowledge of best treatment practices is rapidly evolving, leading to recent publication of multiple international treatment guidelines for HS. SUMMARY: This review compares international HS treatment guidelines, describes evidence for effectiveness of common and emerging HS therapies, and provides guidance for integrating evidence-based HS care into practice. Although over 50 medical and procedural treatments are mentioned across international HS guidelines, only adalimumab and infliximab have grade B/weak recommendation or higher across all major guidelines. This review describes appropriate patient selection and effectiveness of the most commonly used medical and procedural treatments for HS. It also includes recommendations for counseling, dosing, and duration of medical therapies as well as procedure videos for the practicing dermatologist. KEY MESSAGES: Few HS treatments outside of adalimumab are currently supported by high quality randomized clinical trials, contributing to divergence in international treatment guidelines. This article reviews evidence for implementing best practices in HS treatment in the general dermatology clinic.

There are many kidney diseases that might be addressed by gene therapy. However, gene delivery to kidney cells is inefficient. This is due, in part, to the fact that the kidney excludes molecules above 50 kDa and that most gene delivery vectors are megaDaltons in mass. We compared the ability of adeno-associated virus (AAV), adenovirus (Ad), and lentiviral (LV) vectors to deliver genes to renal cells. When vectors were delivered by the intravenous (IV) route in mice, weak luciferase activity was observed in the kidney with substantially more in the liver. When gene delivery was observed in the kidney, expression was primarily in the glomerulus. To avoid these limitations, vectors were injected directly into the kidney by retrograde ureteral (RU) and subcapsular (SC) injections in mice. Small AAV vectors transduced the kidney, but also leaked from the organ and mediated higher levels of transduction in off-target tissues. Comparison of AAV2, 6.2, 8, and rh10 vectors by direct kidney injection demonstrated highest delivery by AAV6.2 and 8. Larger Ad and LV vectors transduced kidney cells and mediated less off-target tissue transduction. These data demonstrate the utility of direct kidney injections to circumvent the kidney size exclusion barrier. They also identify the effects of vector size on on-target and off-target transduction. This lays the foundation for the use of different vector platforms for gene therapy of diverse kidney diseases.

Tien V Nguyen,1,* Catherine M Crosby,2,* Gregory J Heller,3 Zachary I Mendel,3 Mary E Barry,1 Michael A Barry1,4,5 1Department of Internal Medicine, Division of Infectious Diseases, 2Virology and Gene Therapy Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, 3Postbaccalaureate Research Education Program, Mayo Clinic Graduate School of Biomedical Sciences, 4Department of Immunology, 5Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA *These authors contributed equally to this work Background: Human species C adenovirus serotype 5 (Ad5) is the archetype oncolytic adenovirus and has been used in the vast majority of preclinical and clinical tests. While Ad5 can be robust, species C Ad6 has lower seroprevalence, side effects, and appears to be more potent as a systemic therapy against a number of tumors than Ad5. Historically, there have only been four species C human adenoviruses: serotypes 1, 2, 5, and 6. More recently a new species C adenovirus, Ad57, was identified. Ad57 is most similar to Ad6 with virtually all variation in their capsid proteins occurring in the hypervariable regions (HVRs) of their hexon proteins. Most adenovirus neutralizing antibodies target the HVRs on adenoviruses. This led us to replace the hexon HVRs in Ad6 with those from Ad57 to create a new virus called Ad657 and explore this novel species C platform’s utility as an oncolytic virus. Methods: The HVR region from Ad57 was synthesized and used to replace the Ad6 HVR region by homologous recombination in bacteria generating a new viral platform that we call Ad657. Replication-competent Ad5, Ad6, and Ad657 were compared in vitro and in vivo for liver damage and oncolytic efficacy against prostate cancers after single intravenous treatment in mice. Results: Ad5, Ad6, and Ad657 had similar in vitro oncolytic activity against human prostate cancer cells. Ad5 provoked the highest level of liver toxicity after intravenous injection and Ad657 caused the least damage in mice. Previous data demonstrated that Ad6 was superior to Ad5 at killing distant subcutaneous prostate cancer tumors in mouse models after a intravenous injection. Given this, Ad657 was compared to the Ad6 benchmark virus by single intravenous injection into mice bearing subcutaneous human DU145 prostate cancers. Under these conditions, Ad657 first infected the liver and then reached distant tumors. Both Ad6 and Ad657 mediated significant delays in tumor growth and extension of survival with Ad6 mediating higher efficacy. Conclusions: These data suggest that Ad657 may have utility as a local or systemic oncolytic virotherapy for prostate cancers. These data also lay the foundation for serotype-switching with oncolytic species C Ads. Keywords: adenovirus, oncolytic, seroprevalence, virome, prostate cancer

In this work, we present a novel microfluidic impedance biosensor chip for trapping both a single and multiple cancer cells and monitoring their response to the anti-cancer drug treatment. By designing different sizes of working microelectrodes together with the V-shaped cell capture structures, a single or multiple cells are trapped on the microelectrodes surfaces. In addition, by utilizing the passive pumping method, cells can be trapped and positioned inside the microchannels without the need of using the outer micro pump or syringe. The impedance change induced by the response of cells to the anticancer drug Cisplatin treatment was successfully recorded. The proposed biosensor chip has a great potential for applications in cancer cell research, drug screening, and quantification of cancer cells from various tumor stages. The results of this study open potential research collaborations about development of cost-effective devices and lab-on-chips for early disease detection, studies of cancerous cells and their response to anti-cancer drugs to optimize cancer treatments, characterisation of mechanical properties of cells, new drug delivery mechanisms, and micro and nano manufacturing.

We previously selected muscle binding peptides 12.51 and 12.52 from "context-specific" phage display libraries for introduction into adenovirus (Ad) vectors. In this work, these peptides were inserted into the hypervariable region (HVR) 5 loop of the Ad5 hexon protein to display 720 peptides per virions. HVR-12.51 and 12.52 increased transduction of C2C12 cells up to 20-fold when compared to unmodified Ad5. 12.51 increased in vivo muscle transduction 2 to 7-fold over unmodified Ad after intramuscular injection in mice and hamsters. 12.52 did not increase muscle transduction. Notably, insertion of 12.51 into the hexon reduced liver transduction 80-fold when compared to unmodified Ad5 after intravenous injection. Increased muscle transduction in mice translated into increased immune responses after gene-based vaccination. These data suggest there are merits to retargeting and detargeting benefits to modifying the hexons of Ads with peptide ligands.

Wintersweet is a woody ornamental plant and has a long history of human cultivation. Few molecular markers have been characterized and remain scant in wintersweet. This study aimed to mine simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) from the transcriptomic database of wintersweet. A total of 3972 SSRs and 97,060 putative SNPs/indels (92,307 SNPs and 4753 indels) were identified in this data set. This study marks the highest number of SSR and SNP markers discovered to date from wintersweet by using transcriptome sequencing data. These identified markers will provide a useful source for molecular genetic studies such as genetic diversity and characterization, association mapping, and map-based gene cloning in wintersweet.

Oncolytic viruses hold promise as “self-amplifying” cancer therapies wherein a virally killed cell can produce thousands of new viral “drugs” that can kill more cancer cells. Oncolytic viruses may also serve to reawaken or initiate immune responses against cancers by virtue of their abilities to lyse cells, liberate cancer antigens, and provide viral DNA and proteins as immune adjuvants. Oncolytics can also be armed with cytokine or checkpoint inhibitors to amplify antitumor immune responses. Adenoviruses (Ads) are one family of oncolytic viruses. Most human studies have used human Ad serotype 5 (Ad5). Unfortunately, most patients are already immune to Ad5, increasing the likelihood that the agent will be neutralized if used as a cancer therapy. In this work, low seroprevalence Ad6 and Ad57 oncolytic viruses were tested as intravenous therapies against distant prostate cancer sites. After prostate cell infection in vitro, Ad viruses executed these cells efficiently and generated 10,000 to 100,000 progeny infectious viruses. This genome replication also amplified transgene protein production by 100 to 1,000-fold when compared to nonreplicating Ad vectors. Ad5, Ad6, and Ad57 were injected intravenously a single time in nude mice bearing human prostate tumors, and toxicity and efficacy were assessed. Ad6 and 57 produced 30-fold lower liver damage and less toxicity than Ad5. Ad6 and Ad57 significantly repressed the growth of androgen-resistant human DU145 prostate tumors and androgen-sensitive LNCaP prostate tumors and extended survival after single intravenous injection in mice. Ad6 and Ad57 E1 protein were also modified to protect normal cells from viral oncolysis while allowing the virus to amplify and kill prostate cancer cells having perturbations in p300 and pRB pathways. These and other data suggest that targeted and untargeted low seroprevalent Ad6 and Ad57 have better efficacy and safety than the benchmark oncolytic virus Ad5 for systemic therapy of advanced prostate cancer. Citation Format: Tien Nguyen, Michael A. Barry. Systemic oncolytic virotherapy for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A062.

We report the development of a gene replacement strategy for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. VLCAD is a mitochondrial enzyme involved in fatty acid beta-oxidation, a key step in energy production during times of fasting or stress. Deficiency of VLCAD classically presents as hepatic dysfunction, hypoglycemia, cardiomyopathy, rhabdomyolysis, and/or sudden death. While dietary therapy for VLCAD deficiency has proven beneficial in preventing some symptoms, a risk of metabolic catastrophic decompensation remains throughout life during times of increased energy demand. We designed a recombinant adeno-associated virus (AAV) expressing the human VLCAD gene (AAV8-hVLCAD). To demonstrate its in vivo activity, AAV8-hVLCAD was administered via the tail vein to VLCAD-knockout mice. A reduction in accumulated serum long-chain acylcarnitines and increased fasting tolerance judged on blood glucose concentrations were observed as of 11 days postinjections through100 days. Western analysis of liver, skeletal muscle, and heart extracts using PEP1 anti-hVLCAD antibody revealed short-term hVLCAD expression in the liver and muscle and longer-term expression in heart. This demonstrates the ability of human VLCAD to correct the biochemical phenotype of VLCAD-deficient mice.

Importance Effective patient-physician communication is essential for optimal health care. Recent introduction of online patient portals to access test results are changing the communication landscape, but regulatory guidelines for the online release of biopsy results vary from state to state. Objectives To assess patient preferences for receiving skin biopsy results to rule out melanoma and to compare those preferences to current physicians’ practices for notification. Design, Setting, and Participants English-speaking individuals 18 years or older were recruited consecutively from melanoma clinics at 3 academic tertiary referral medical centers: University of California, San Francisco, University of Pennsylvania, and Duke University. Patients were surveyed from July 1, 2012, through July 31, 2013. A second survey of physicians at these institutions was conducted to assess physician notification practices. Results A total of 301 of 305 patients agreed to participate (98.7 response rate). Most of the patients (67.1%) preferred to speak directly with their physician by telephone to receive their skin biopsy results, followed by a distant second choice (19.5%) of being notified in person at a clinic visit. Voice message or online patient portal were each the preferred method of communication for 5.1% of patients. The most important consideration for patients was a communication modality that provided test results in the most rapid manner; 51.7% wanted a method that was rapid, and 7.8% preferred a method that was not only speedy but also allowed them an opportunity to ask questions. A total of 59.5% of the study participants would choose the same communication method regardless of the biopsy results, but 40.5% preferred a different mode of notification if their biopsy results revealed a malignant tumor. Younger and more highly educated patients favored the online portal. Of 84 physicians surveyed, 47 responded (56% response rate). Physicians’ overall preferred method of contacting patients aligned with patient preference for speaking by telephone (56.5%). However, for benign results, 31.2% of physicians chose to speak by telephone, whereas patients preferred voicemail (32.1%). There was physician uncertainty as to guidelines regarding communication of test results. Conclusions and Relevance Patient preference has shifted from face-to-face visit to discussion over the telephone because of a desire for rapid notification. Experience with online portal delivery of results favorably inclined patients toward that modality. We recommend that patients be queried regarding their notification preference on the biopsy consent form.

The acyl-CoA dehydrogenases are a family of mitochondrial flavoproteins involved in the catabolism of fatty and amino acids. Isobutyryl-CoA dehydrogenase (IBD) is involved in the catabolism of valine and catalyzes the conversion of isobutyryl-CoA to methacrylyl-CoA. The crystal structure of IBD with and without substrate has been determined to 1.76-A resolution. The asymmetric unit contains a homotetramer with substrate/product bound in two monomers. The overall structure of IBD is similar to those of previously determined acyl-CoA dehydrogenases and consists of an NH2-terminal α-helical domain, a medial β-strand domain and a C-terminal α-helical domain. The enzyme-bound ligand has been modeled in as the reaction product, methacrylyl-CoA. The location of Glu-376 with respect to the C-2–C-3 of the bound product and FAD confirms Glu-376 to be the catalytic base. IBD has a shorter and wider substrate-binding cavity relative to short-chain acyl-CoA dehydrogenase, permitting the optimal binding of the isobutyryl-CoA substrate. The dramatic lateral expansion of the binding cavity seen in isovaleryl-CoA dehydrogenase is not observed in IBD. The conserved tyrosine or phenylalanine that defines a side of the binding cavity in other acyl-CoA dehydrogenases is replaced by a leucine (Leu-375) in the current structure. Substrate binding changes the position of some residues lining the binding pocket as well as the position of the loop containing the catalytic glutamate and subsequent helix. Three clinical mutations have been modeled to the structure. The mutations do not affect substrate binding but instead appear to disrupt protein folding and/or stability.

The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes that catalyze the alpha,beta-dehydrogenation of acyl-CoA esters. Two homologues active in branched chain amino acid metabolism have previously been identified. We have used expression in Escherichia coli to produce a previously uncharacterized ACD-like sequence (ACAD8) and define its substrate specificity. Purified recombinant enzyme had a k(cat)/K(m) of 0.8, 0.23, and 0.04 (microM(-1)s(-1)) with isobutyryl-CoA, (S) 2-methylbutyryl-CoA, and n-propionyl-CoA, respectively, as substrates. Thus, this enzyme is an isobutyryl-CoA dehydrogenase. A single patient has previously been described whose fibroblasts exhibit a specific deficit in the oxidation of valine. Amplified ACAD8 cDNA made from patient fibroblast mRNA was homozygous for a single nucleotide change (905GA) in the ACAD8 coding region compared to the sequence from control cells. This encodes an Arg302Gln substitution in the full-length protein (position 280 in the mature protein), a position predicted by molecular modeling to be important in subunit interactions. The mutant enzyme was stable but inactive when expressed in E. coli. It was also stable and appropriately targeted to mitochondria, but inactive when expressed in mammalian cells. These data confirm further the presence of a separated ACD in humans specific to valine catabolism (isobutyryl-CoA dehydrogenase, IBDH), along with the first enzymatic and molecular confirmation of a deficiency of this enzyme in a patient.

Transition-metal catalysis is out-grown from dry-boxes where the use of inert gas atmosphere and the exclusion of moisture have been essential. Such a restriction undoubtedly imposes limitations in the application of these reactions in organic synthesis and in the recycling of the catalysts. This article discusses some recent advances of rhodium-catalyzed carbon­carbon bond formations under the natural conditions of air and water.

Autoinflammation is characterized by aberrant regulation of the innate immune system and often manifests as periodic fevers and systemic inflammation involving multiple organs, including the skin. Mutations leading to abnormal behavior or activity of the interleukin 1 beta (IL-1s)-processing inflammasome complex have been found in several rare autoinflammatory syndromes, for which anticytokine therapy such as IL-1 or tumor necrosis factor-alfa inhibition may be effective. It is becoming clear that features of autoinflammation also affect common dermatoses, some of which were previously thought to be solely autoimmune in origin (eg, vitiligo, systemic lupus erythematosus). Recognizing the pathogenetic role of autoinflammation can open up new avenues for the targeted treatment of complex, inflammatory dermatoses.

Recent genome-wide association studies (GWAS) have identified multiple genetic risk factors for psoriasis, but data on their association with age of onset have been marginally explored. The goal of this study was to evaluate known risk alleles of psoriasis for association with age of psoriasis onset in three well-defined case-only cohorts totaling 1,498 psoriasis patients. We selected 39 genetic variants from psoriasis GWAS and tested these variants for association with age of psoriasis onset in a meta-analysis. We found that rs10484554 and rs12191877 near HLA-C and rs17716942 near IFIH1 were associated with age of psoriasis onset with false discovery rate < 0.05. The association between rs17716942 and age of onset was not replicated in a fourth independent cohort of 489 patients (). The imputed HLA-C*06:02 allele demonstrated a much stronger association with age of psoriasis onset than rs10484554 and rs12191877. We conclude that despite the discovery of numerous psoriasis risk alleles, HLA-C*06:02 still plays the most important role in determining the age of onset of psoriasis. Larger studies are needed to evaluate the contribution of other risk alleles, including IFIH1, to age of psoriasis onset.

Psoriasis is a chronic, inflammatory skin condition with negative impacts both physical and psychological. Scalp psoriasis, especially around the hairline, can cause significant impairment in quality of life due to its visibility. Options for treatment of facial psoriasis, including hairline involvement, are the use of low potency topical steroids, calcineurin inhibitors, and vitamin D analogues. Though the use of excimer laser for scalp psoriasis has been reported, there are no cases or studies specifically examining excimer laser phototherapy for the treatment of hairline psoriasis. We present a case of rapid improvement of hairline psoriasis using a regimen of 308 nm excimer laser with clobetasol spray and recommend an algorithm for the optimal treatment of scalp psoriasis utilizing currently available antipsoriatic therapies.

Thiopurine S -methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT genetic polymorphisms represent a striking example of the potential clinical value of pharmacogenetics. Subjects homozygous for TPMT * 3A , the most common variant allele for low activity, an allele that encodes a protein with two changes in amino acid sequence, are at greatly increased risk for life-threatening toxicity when treated with standard doses of thiopurines. These subjects have virtually undetectable levels of TPMT protein. In this study, we tested the hypothesis that TPMT * 3A might result in protein misfolding and aggregation. We observed that TPMT * 3A forms aggresomes in cultured cells and that it aggregates in vitro , functional mechanisms not previously described in pharmacogenetics. Furthermore, there was a correlation among TPMT half-life values in rabbit reticulocyte lysate, aggresome formation in COS-1 cells, and protein aggregation in vitro for the three variant allozymes encoded by alleles that include the two TPMT * 3A single-nucleotide polymorphisms. These observations were compatible with a common structural explanation for all of these effects, a conclusion supported by size-exclusion chromatography and CD spectroscopy. The results of these experiments provide insight into a unique pharmacogenetic mechanism by which common polymorphisms affect TPMT protein function and, as a result, therapeutic response to thiopurine drugs.

The acyl-CoA dehydrogenases are a family of mitochondrial flavoproteins involved in the catabolism of fatty and amino acids. Isobutyryl-CoA dehydrogenase (IBD) is involved in the catabolism of valine and catalyzes the conversion of isobutyryl-CoA to methacrylyl-CoA. The crystal structure of IBD with and without substrate has been determined to 1.76-A resolution. The asymmetric unit contains a homotetramer with substrate/product bound in two monomers. The overall structure of IBD is similar to those of previously determined acyl-CoA dehydrogenases and consists of an NH2-terminal alpha-helical domain, a medial beta-strand domain and a C-terminal alpha-helical domain. The enzyme-bound ligand has been modeled in as the reaction product, methacrylyl-CoA. The location of Glu-376 with respect to the C-2-C-3 of the bound product and FAD confirms Glu-376 to be the catalytic base. IBD has a shorter and wider substrate-binding cavity relative to short-chain acyl-CoA dehydrogenase, permitting the optimal binding of the isobutyryl-CoA substrate. The dramatic lateral expansion of the binding cavity seen in isovaleryl-CoA dehydrogenase is not observed in IBD. The conserved tyrosine or phenylalanine that defines a side of the binding cavity in other acyl-CoA dehydrogenases is replaced by a leucine (Leu-375) in the current structure. Substrate binding changes the position of some residues lining the binding pocket as well as the position of the loop containing the catalytic glutamate and subsequent helix. Three clinical mutations have been modeled to the structure. The mutations do not affect substrate binding but instead appear to disrupt protein folding and/or stability.

Background & Aims: Diabetic gastroparesis (delayed gastric emptying) is a well-recognized complication of diabetes that causes considerable morbidity and makes glucose control difficult. Interstitial cells of Cajal, which express the receptor tyrosine kinase Kit, are required for normal gastric emptying. We proposed that Kit expression is lost during diabetic gastroparesis due to increased levels of oxidative stress caused by low levels of heme oxygenase-1 (HO-1), an important cytoprotective molecule against oxidative injury. Methods: Gastric emptying was measured in nonobese diabetic mice and correlated with levels of HO-1 expression and activity. Endogenous HO-1 activity was increased by administration of hemin and inhibited by chromium mesoporphyrin. Results: In early stages of diabetes, HO-1 was up-regulated in gastric macrophages and remained up-regulated in all mice that were resistant to development of delayed gastric emptying. In contrast, HO-1 did not remain up-regulated in all the mice that developed delayed gastric emptying; expression of Kit and neuronal nitric oxide synthase decreased markedly in these mice. Loss of HO-1 up-regulation increased levels of reactive oxygen species. Induction of HO-1 by hemin decreased reactive oxygen species, rapidly restored Kit and neuronal nitric oxide synthase expression, and completely normalized gastric emptying in all mice. Inhibition of HO-1 activity in mice with normal gastric emptying caused a loss of Kit expression and development of diabetic gastroparesis. Conclusions: Induction of the HO-1 pathway prevents and reverses cellular changes that lead to development of gastrointestinal complications of diabetes. Reagents that induce this pathway might therefore be developed as therapeutics.

Cardiomyopathy is an important cause of morbidity and mortality in children. Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD), one of several nuclear encoded mitochondrial enzymes that catalyze the initial step in the beta-oxidation of straight chain fatty acids, often presents with cardiomyopathy and/or sudden death. How VLCAD deficiency causes cardiomyopathy is unclear, but appears to be related to the accumulation of toxic long-chain acyl-carnitine species rather than a block in energy metabolism. A recent report of a 5-year-old with VLCAD deficiency note resolution of her hypertrophic cardiomyopathy while on a strict low fat diet supplemented with medium chain triglycerides and carnitine. While dietary therapy for VLCAD deficiency has been beneficial to some, many people to not respond. Intercurrent illness with diminished oral intake, exercise, or fasting all increase demand for fatty acid metabolism and can potentially trigger catastrophic events even while on dietary therapy.