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To determine the epidemiology of human parainfluenza virus in homeless shelters during the COVID-19 pandemic, we analyzed data and sequences from respiratory specimens collected in 23 shelters in Washington, USA, during 2019-2021. Two clusters in children were genetically similar by shelter of origin. Shelter-specific interventions are needed to reduce these infections.

Background Rhinovirus (RV) is a common cause of respiratory illness in all people, including those experiencing homelessness. RV epidemiology in homeless shelters is unknown. Methods We analyzed data from a cross-sectional homeless shelter study in King County, Washington, October 2019–May 2021. Shelter residents or guardians aged ≥3 months reporting acute respiratory illness completed questionnaires and submitted nasal swabs. After 1 April 2020, enrollment expanded to residents and staff regardless of symptoms. Samples were tested by multiplex RT-PCR for respiratory viruses. A subset of RV-positive samples was sequenced. Results There were 1066 RV-positive samples with RV present every month of the study period. RV was the most common virus before and during the coronavirus disease 2019 (COVID-19) pandemic (43% and 77% of virus-positive samples, respectively). Participants from family shelters had the highest prevalence of RV. Among 131 sequenced samples, 33 RV serotypes were identified with each serotype detected for ≤4 months. Conclusions RV infections persisted through community mitigation measures and were most prevalent in shelters housing families. Sequencing showed a diversity of circulating RV serotypes, each detected over short periods of time. Community-based surveillance in congregate settings is important to characterize respiratory viral infections during and after the COVID-19 pandemic. Clinical Trials Registration NCT04141917.

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.

Mitigation measures against the COVID-19 pandemic affected the RSV seasonality and led to an unexpectedly high number of RSV cases in Washington State since October 2022. Here we describe the RSV genomic characteristics and evolutionary relationship of 2022 outbreak compared to the previous RSV outbreaks in the region and globally.

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.

The circulation of respiratory viruses poses a significant health risk among those residing in congregate settings. Data are limited on seasonal human coronavirus (HCoV) infections in homeless shelter settings.We analysed data from a clinical trial and SARS-CoV-2 surveillance study at 23 homeless shelter sites in King County, Washington between October 2019-May 2021. Eligible participants were shelter residents aged ≥3 months with acute respiratory illness. We collected enrolment data and nasal samples for respiratory virus testing using multiplex RT-PCR platform including HCoV. Beginning April 1, 2020, eligibility expanded to shelter residents and staff regardless of symptoms. HCoV species was determined by RT-PCR with species-specific primers, OpenArray assay or genomic sequencing for samples with an OpenArray relative cycle threshold22.Of the 14,464 samples from 3281 participants between October 2019-May 2021, 107 were positive for HCoV from 90 participants (median age 40 years, range: 0·9-81 years, 38% female). HCoV-HKU1 was the most common species identified before and after community-wide mitigation. No HCoV-positive samples were identified between May 2020-December 2020. Adults aged ≥50 years had the highest detection of HCoV (11%) among virus-positive samples among all age-groups. Species and sequence data showed diversity between and within HCoV species over the study period.HCoV infections occurred in all congregate homeless shelter site age-groups with the greatest proportion among those aged ≥50 years. Species and sequencing data highlight the complexity of HCoV epidemiology within and between shelters sites.Gates Ventures, Centers for Disease Control and Prevention, National Institute of Health.

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.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease that severely impairs patients' quality of life. It is characterized by recurrent painful nodules, abscesses and draining sinus tracts in primarily intertriginous areas. We aimed to review the most up-to-date information regarding the epidemiology, clinical presentation, diagnostic studies, pathogenesis, comorbidities and quality of life of patients with hidradenitis suppurativa. We performed a systematic search of Medline, Embase database (from inception to September 2019) and review of bibliographies without restrictions on year or language. HS has an estimated global prevalence of 0.00033-4.1% (but most likely 0.7-1.2% in the European-US population). Patients still experience a significant diagnostic delay, up to several years. In the absence of pathognomonic tests, the diagnosis of HS is made from clinical observation and the disease narrative. Phenotypic variation renders diagnosis and severity assessment difficult. Ultrasound imaging is an emerging assessment tool for deep-seated lesions. The Hurley Staging System is still widely used in severity rating. Follicular hyperkeratosis and dilatation, follicular rupture and chronic inflammation with architectural tissue changes have been implicated in the pathogenesis of HS. HS has been associated with metabolic syndrome and other risk factors for cardiovascular disease, diabetes mellitus type II, polycystic ovarian syndrome, depression, suicide and substance use disorders. It has been linked to other immune-mediated diseases such as inflammatory bowel disease and spondyloarthropathy. Pain, pruritus, malodour, low self-esteem, sleep and sexual dysfunctions, and poor mental health are chronic symptoms or consequences of uncontrolled disease. HS is an under-diagnosed and under-treated disease with a profound negative impact on patients' quality of life. In the light of its associated comorbidities, an interdisciplinary management approach may be needed to ensure the best outcomes.

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.

The phenomenon whereby phototherapy exacerbates instead of reduces intensely inflamed skin may be known to most, but not all, phototherapy providers. Furthermore, it has never been articulated or named in the literature. We report a patient with near-erythrodermic psoriatic skin which became fully erythrodermic after one treatment with narrowband UVB, and the results of a survey study disseminated to all phototherapy practitioners registered with the National Psoriasis Foundation. We propose the name “hyper-photosensitivity threshold” to refer to the aforementioned phenomenon. Fifty-six percent of our survey respondents reported having observed this phenomenon and the following “cooling” procedures to reduce the intense inflammation of erythrodermic skin: topical corticosteroids, cyclosporine, wet wraps, and systemic medications other than cyclosporine, ranging from systemic steroids and methotrexate to mycophenolate mofetil. We hope that this knowledge will help future generations of phototherapy practitioners treat intensely inflamed skin properly and avoid unwanted adverse effects.

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.

Background Hidradenitis suppurativa (HS) is a chronic inflammatory disorder characterized by sterile abscesses and fistulae predominantly affecting the axillae and groin. Various biologic agents have been attempted for HS, but there is still no definitive treatment. Objectives We sought to evaluate the efficacy, safety, and tolerability of anakinra in the treatment of moderate to severe HS. Methods Six patients with moderate to severe HS were enrolled in an open-label study with all patients receiving active treatment for 8 weeks with an additional 8 weeks of follow-up off therapy. Results The 5 patients who completed the 8-week therapy showed a significant mean decrease in their modified Sartorius score of 34.8 points. The physician and patient global assessment of overall activity showed significant reductions between baseline and 8 weeks of therapy: 45.8 points and 35.6 points, respectively. The Dermatology Life Quality Index showed a significant reduction after 8 weeks of treatment with anakinra. Functional T-cell analysis revealed that patients had increased percentages of CD3 + T cells in lesional skin compared with nonlesional skin before therapy. Limitations The limited number of patients and lack of control group are limitations. Conclusions Anakinra demonstrated decreased HS disease activity by both objective and subjective measures.

Psoriasis is a chronic skin condition that requires long-term maintenance therapy, sometimes lifelong. Phototherapy (narrowband ultraviolet B, broadband ultraviolet B, and psoralen plus ultraviolet A) is a safe and effective method of treating generalized psoriasis. Although there are numerous publications demonstrating the efficacy of phototherapy for the initial clearance of psoriasis, there has not been a review evaluating the merits of phototherapy for the maintenance of remission in patients with psoriasis once they clear their skin disease. We searched PubMed for articles evaluating the efficacy of phototherapy for maintaining remission in patients with treated generalized psoriasis. The majority of published trials supported the use of maintenance phototherapy after clearance of generalized psoriasis. Typically, the studies showed that maintenance phototherapy reduces the rate of disease relapse and prolongs the duration of remission of psoriasis after initial clearance. There is available evidence supporting phototherapy, including psoralen plus ultraviolet A, as a safe and effective treatment option for maintenance of remission in patients with generalized psoriasis. Further studies are needed to adequately assess narrowband ultraviolet B for maintenance therapy, and additional studies are also necessary to directly compare the efficacy of different phototherapy types for remission maintenance.

The negative impact of psoriasis on a patient's quality of life (QoL) is well documented in the literature. Patients often suffer poor self-esteem, difficulties in social interactions, and significant psychological distress. It is, therefore, critically important that a clinician evaluate the extent to which the disease impacts a patient's QoL. This chapter reviews several validated and reliable generic, dermatology-specific, and disease-specific QoL instruments useful in measuring the impact of psoriasis on patient's QoL. These QoL instruments can be especially helpful in identifying those patients who would most benefit from systemic or biologic therapy.

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.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder of fatty acid beta-oxidation that can present at any age with cardiomyopathy, rhabdomyolysis, hepatic dysfunction, and/or nonketotic hypoglycemia. Through the expansion of newborn screening programs an increasing number of individuals with VLCAD deficiency are being identified prior to the onset of symptoms allowing early initiation of therapy. The development of a safe, durable, and effective VLCAD gene delivery system for use at the time of diagnosis could result in a significant improvement in the quality and duration of life for patients with VLCAD deficiency. To this end, we developed a construct containing the human VLCAD cDNA under the control of the strong CMV promoter (pCMV-hVLCAD). A novel rabbit polyclonal anti-VLCAD antibody was prepared using a 24 amino-acid peptide unique to the human VLCAD protein to study human VLCAD expression in immune competent mice. Antibody specificity was demonstrated in Western blots of human VLCAD deficient fibroblasts and in pCMV-hVLCAD transiently transfected VLCAD deficient fibroblasts. Transfected fibroblasts showed correction of the metabolic block as demonstrated by normalization of C14- and C16-acylcarnitine species in cell culture media and restoration of VLCAD activity in cells. Following tail vein injection of pCMV-hVLCAD into mice, we demonstrated expression of hVLCAD in liver. Altogether, these steps are important in the development of a durable gene therapy for VLCAD deficiency.

Short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle with optimal activity toward butyryl- and hexanoyl-CoA. Two common variants of this enzyme encoding G185S and R147W substitutions have been identified at an increased frequency compared to the general population in patients with a wide variety of clinical problems, but functional studies of the purified mutant enzymes have shown only modestly changed kinetic properties. Moreover, both amino acid residues are located quite far from the catalytic pocket and the essential FAD cofactor. To clarify the potential relationship of these variants to clinical disease, we have further investigated their thermodynamic properties using spectroscopic and electrochemical techniques. Purified R147W hSCAD exhibited almost identical physical and redox properties to wild-type but only half of the specific activity and substrate activation shifts observed in wild-type enzyme. In contrast, the G185S mutant proved to have impairments of both its kinetic and electron transfer properties. Spectroelectrochemical studies reveal that G185S binding to the substrate/product couple produces an enzyme potential shift of only +88 mV, which is not enough to make the reaction thermodynamically favorable. For wild-type hSCAD, this barrier is overcome by a negative shift in the substrate/product couple midpoint potential, but in G185S this activation was not observed. When G185S was substrate bound, the midpoint potential of the enzyme actually shifted more negative. These results provide valuable insight into the mechanistic basis for dysfunction of the common variant hSCADs and demonstrate that mutations, regardless of their position in the protein structure, can have a large impact on the redox properties of the enzyme.

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.

Ms. Austin is a healthy 21 year-old woman, who appears very anxious in the waiting room. She has a 2-year history of acne vulgaris unresponsive to herbal remedies, topical retinoid agents, and combined topical antibiotic/benzoyl peroxide preparations. She reports that within the past 3 months she has suffered significant discomfort and public embarrassment from the inflamed deep cysts. She is here to get a second opinion about how to improve her acne, stating that her general practitioner has done everything in his power to help her.

Mr. Adams is 27 year-old Caucasian new patient, who presented to the dermatology clinic with several excoriations on the face, arms, and legs. The lesions were all located on easily reachable areas of his anatomy such as the extensor arms but not the medial arms, the anterior legs but not the posterior legs, etc. No primary skin lesions were seen. The patient was visibly depressed. However, when asked what was causing him to be distressed, he answered: “nothing other than having these spots on my [his] skin.” The practitioner, sensing that forward questions might not be useful, decided to change the questioning approach and asked about his general wellbeing. The patient then reported severe early insomnia (i.e., he cannot fall asleep), middle insomnia (i.e., he wakes up frequently in the middle of the night), and terminal insomnia (i.e., he wakes up early in the morning and cannot go back to sleep even despite being extremely tired).

Mr. Hemingway is 45-year-old business executive with a very busy work schedule. He was seen in your office yesterday morning for a concerning flat, uniformly brown mole that had been expanding from 3 to 8 mm in diameter and darkening for 6 months. A 3-mm punch biopsy of the lesion was performed, and you distinctly remember having placed the skin specimen in its properly labeled container before sending it to the pathology laboratory. This morning, you received a message from the administrative assistant in the lab stating that no specimen was found in the container for Mr. Hemingway. You called the manager of the lab and asked for help. Sadly, after an exhaustive search of the lab, Mr. Hemingway’s specimen still could not be found. You also performed a similar search without finding the specimen.

Mrs. Aaron is a 56 year-old African-American woman, who presented with a chief complaint of a constant sensation of being covered by “cobwebs” on her face for the past 8 months. She did not seen any spiders or insects nearby when this sensation occurred, and appeared unconcerned about the possibility of an infestation. Upon examination, she showed no primary skin lesions but was observed to wipe her face with her hand frequently as if to get rid of these cobwebs. She related that due to the intensity of the cobweb sensation, she had not been able to focus on any task – which had hindered her from gaining employment since the start of her disease.

Ms. Reed is a 45 year-old woman with a history of chronic atopic dermatitis, which started when she was in college. At first, she reportedly experienced episodes of patchy, itchy red bumps on the extensor aspects of her arms, legs, and such episodes would get worse during exam periods. After college, she had ten blissful years without any significant atopic dermatitis episodes. However, the disease has been making a return in the past 12–13 years, gradually at first but then picking up speed in terms of frequency and viciousness of each episode. Today, the patient comes in with both arms and legs studded circumferentially with eczematous papules, which appear beefy red and noticeably warm to the touch. She reports still using her triamcinolone acetonide 0.1 % cream and emollients to the affected areas daily. You have never observed such inflammatory intensity with her skin disease before.

Ms. Dickinson is a 56 year-old new patient presenting with severe scalp dermatitis and psoriasiform scaling, for which a tentative diagnosis of scalp psoriasis has been made. She reports trying several topical agents, each for a few days on six different occasions over a 3-month period, with inadequate control. The referring dermatologist, who called you prior to the patient visit, is concerned that her insufficient response to therapy has been a result of poor compliance. He notes that she typically resists his treatment recommendations, and on several occasions has made blanket statements indicating her distrust of medical professionals.

Mr. Twain is a 46 year-old man with hidradenitis suppurativa that has not been active for the last year. At his last follow-up 3 months ago, he reported groin pain in spite of a benign physical exam. Today his chief concern is again pain: “Doctor, I have been aching in my armpits a lot recently. I haven’t seen any boils and bumps in these areas, but I keep feeling the pain. It’s worse at night when I try to go to sleep.”

Mr. Schmidt is a 38 year-old Caucasian man presenting with a 3-month history of extremely bothersome crawling, biting, and stinging sensations. He reported that at times he felt as if “a 1,000 bugs were falling and crawling on my [his] skin.” The patient claimed that no living organisms were involved in causing his skin condition, not only because there was no evidence to suggest this but also because the symptomatology was so bizarre that he knew of no organisms likely to cause this type of discomfort. The onset of these symptoms was not correlated with any particular events or stressors, and he denied recreational drug use. Past medical history was unremarkable. Due to the intensity of the formication, he was very eager to undergo aggressive therapy to reduce these sensations.

Mrs. Hightower is a 79-year-old African-American female, who presents to the office with her husband with a chief complaint of persistent bug bites and edema of the lower extremities. The lesions in question are reported on both arms from the wrist to mid upper arm, and the lower extremities from just proximal to the toes to below the knee. The extremities appear erythematous, swollen, and scaly with multiple vesicles and areas denuded of overlying skin. The patient is insistent that the cause of her discomfort is parasites. She claims to have had this complaint constantly for 6 years, starting just after a quadruple bypass and continuing without interruption till the current visit. She reports a history of kidney failure, fatigue, and fluid retention, but denies any other symptoms.