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Horizon scanning is intended to identify the opportunities and threats associated with technological, regulatory and social change. In 2017 some of the present authors conducted a horizon scan for bioengineering (Wintle et al., 2017). Here we report the results of a new horizon scan that is based on inputs from a larger and more international group of 38 participants. The final list of 20 issues includes topics spanning from the political (the regulation of genomic data, increased philanthropic funding and malicious uses of neurochemicals) to the environmental (crops for changing climates and agricultural gene drives). The early identification of such issues is relevant to researchers, policy-makers and the wider public.

Advances in biological engineering are likely to have substantial impacts on global society. To explore these potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunities and risks presented by biological engineering. We first identified 70 potential issues, and then used an iterative process to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to be relatively unknown outside the field of biological engineering. The issues identified may be of interest to researchers, businesses and policy makers in sectors such as health, energy, agriculture and the environment.

Recognizing that certain biological functions can be associated with specific DNA sequences has led various fields of biology to adopt the notion of the genetic part. This concept provides a finer level of granularity than the traditional notion of the gene. However, a method of formally relating how a set of parts relates to a function has not yet emerged. Synthetic biology both demands such a formalism and provides an ideal setting for testing hypotheses about relationships between DNA sequences and phenotypes beyond the gene-centric methods used in genetics. Attribute grammars are used in computer science to translate the text of a program source code into the computational operations it represents. By associating attributes with parts, modifying the value of these attributes using rules that describe the structure of DNA sequences, and using a multi-pass compilation process, it is possible to translate DNA sequences into molecular interaction network models. These capabilities are illustrated by simple example grammars expressing how gene expression rates are dependent upon single or multiple parts. The translation process is validated by systematically generating, translating, and simulating the phenotype of all the sequences in the design space generated by a small library of genetic parts. Attribute grammars represent a flexible framework connecting parts with models of biological function. They will be instrumental for building mathematical models of libraries of genetic constructs synthesized to characterize the function of genetic parts. This formalism is also expected to provide a solid foundation for the development of computer assisted design applications for synthetic biology.

Introduction Socio-economic and political events of recent years have caused a significant increase in immigrants attempting to illegally cross the United States (US)-Mexico border. While a 30-foot border wall separates the US and Mexico, immigrants from around the world have used this location as their point of entry to the US. These border crossings have led to a dramatic increase in major trauma resulting in increased inpatient resource utilization and the need for comprehensive hospital services. The aim of this study was to describe the nationality of injured immigrants admitted to a Trauma Center serving a segment of the US-Mexico border wall and to report their ultimate destinations after discharge.Methods We performed a retrospective review of patients admitted to an academic, Level 1 Trauma Center after injury at the US-Mexico border wall from 2021 to 2022. Demographic information was obtained from the trauma registry. The electronic medical record was searched to identify each patient’s self-reported country of origin. Patients’ nationality was then stratified by region of the world to understand geographic representation of border injury admissions.Results We identified 597 patients injured while crossing the US-Mexico border wall representing 38 different countries. The mean age of patients was 32.2±10.4 years and 446 (75%) were male. Most patients (405, 67.8%) were Mexican, followed by 23 (3.9%) patients from Peru, 17 (2.8%) patients from India, 14 (2.3%) patients from El Salvador, 13 (2.2%) patients from Cuba and 12 (2.0%) patients from Jamaica. When considering regions of the world other than Mexico, patients were most commonly from Africa, South America and Central America.Conclusion The increased volume of trauma associated with the US-Mexico border wall is a humanitarian and health crisis.(1) The diverse national origin of patients admitted after injury from border wall falls has shed new light on the social and interpreter services needed to care for these border injury patients and the challenges that exist in their post-discharge care.

Abstract Extracellular vesicles (EVs) are integral to numerous biological processes, yet it is unclear how environmental factors or interactions among individuals within a population affect EV-regulated systems. In Caenorhabditis elegans, the evolutionarily conserved large EVs, known as exophers, are part of a maternal somatic tissue resource management system. Consequently, the offspring of individuals exhibiting active exopher biogenesis (exophergenesis) develop faster. Our research focuses on unraveling the complex inter-tissue and social dynamics that govern exophergenesis. We found that ascr#10, the primary male pheromone, enhances exopher production in hermaphrodites, mediated by the G-protein-coupled receptor STR-173 in ASK sensory neurons. In contrast, pheromone produced by other hermaphrodites, ascr#3, diminishes exophergenesis within the population. This process is regulated via the neuropeptides FLP-8 and FLP-21, which originate from the URX and AQR/PQR/URX neurons, respectively. Our results reveal a regulatory network that controls the production of somatic EV by the nervous system in response to social signals.

Introduction Direct oral anticoagulant (DOAC) use is becoming more prevalent in patients presenting after trauma. We sought to identify the prevalence and predictors of subtherapeutic and therapeutic DOAC concentrations and hypothesized that increased anti-Xa levels would correlate with increased risk of bleeding and other poor outcomes.Methods A retrospective cohort study of all trauma patients on apixaban or rivaroxaban admitted to a level 1 trauma center between January 2015 and July 2021 was performed. Patients were excluded if they did not have a DOAC-specific anti-Xa level at presentation. Therapeutic levels were defined as an anti-Xa of 50 ng/mL to 250 ng/mL for rivaroxaban and 75 ng/mL to 250 ng/mL for apixaban. Linear regression was used to identify correlations between study variables and anti-Xa level, and binomial logistic regression was used to test the association of anti-Xa level with outcomes.Results There were 364 trauma patients admitted during the study period who were documented to be on apixaban or rivaroxaban. Of these, 245 patients had anti-Xa levels measured at admission. The population was 53% woman, with median age of 78 years, and median Injury Severity Score of 5. In total, 39% of patients had therapeutic and 20% had supratherapeutic anti-Xa levels. Female sex, increased age, decreased height and weight, and lower estimated creatinine clearance were associated with higher anti-Xa levels at admission. There was no correlation between anti-Xa level and the need for transfusion or reversal agent administration, admission diagnosis of intracranial hemorrhage (ICH), progression of ICH, hospital length of stay, or mortality.Conclusions Anti-Xa levels in trauma patients on DOACs vary widely; female patients who are older, smaller, and have decreased kidney function present with higher DOAC-specific anti-Xa levels after trauma. We were unable to detect an association between anti-Xa levels and clinical outcomes.Level of evidence III—Prognostic and Epidemiological.

Abstract Introduction Radiation oncology patient pathways are complex. This complexity creates risk and potential for error to occur. Comprehensive safety and quality management programmes have been developed alongside the use of incident learning systems (ILSs) to mitigate risks and errors reaching patients. Robust ILSs rely on the safety culture (SC) within a department. The aim of this study was to assess perceptions and understanding of SC and ILSs in two closely linked radiation oncology departments and to use the results to consider possible quality improvement (QI) of department ILSs and SC. Methods A survey to assess perceptions of SC and the currently used ILSs was distributed to radiation oncologists, radiation therapists and radiation oncology medical physicists in the two departments. The responses of 95 staff were evaluated (63% of staff). The findings were used to determine any areas for improvement in SC and local ILSs. Results Differences were shown between the professional cohorts. Barriers to current ILS use were indicated by 67% of respondents. Positive SC was shown in each area assessed: 69% indicated the departments practised a no‐blame culture. Barriers identified in one department prompted a QI project to develop a new reporting system and process, improve departmental learning and modify the overall ILS. Conclusion An understanding of SC and attitudes to ILSs has been established and used to improve ILS reporting, feedback on incidents, departmental learning and the QA program. This can be used for future comparisons as the systems develop.

Recent clinical successes have propelled recombinant adeno-associated virus vectors (rAAV) to the center stage for human gene therapy applications. However, the exploding demand for high titers of highly pure rAAV vectors for clinical applications and market needs remains hindered by challenges met at the manufacturing stage. The production of rAAV by transfection in suspension cells remains one of the most commonly used production platforms. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). Further, we detail a new, robust, and highly efficient downstream purification protocol compatible with both transfection and infection-based harvests that generated rAAV9 stocks of high purity. Our in-depth comparison revealed quantitative, qualitative, and biological differences between PEI-mediated transfection and HSV infection. The HSV production system yielded to higher rAAV vector titers, higher specific yields, and a higher percentage of full capsids than transfection. Furthermore, HSV-produced stocks had a significantly lower concentration of residual host cell proteins and helper DNA impurities, but contained detectable levels of HSV DNA. Importantly, the potency of PEI-produced and HSV-produced rAAV stocks were identical. Analyses of AAV Rep and Cap expression levels and replication showed that HSV-mediated production led to a lower expression of Rep and Cap, but increased levels of AAV genome replication. Our methodology enables high-yield, high purity rAAV production and a biological framework to improve transfection quality and yields by mimicking HSV-induced biological outcomes.

Coxiella burnetii, the causative bacterium of the zoonotic disease Q fever, has been documented in many different species. We describe documented turtles that were PCR positive for C. burnetii from multiple locations in Illinois and Wisconsin, USA. Assessing the conservation implications, reservoir potential, and zoonotic risk requires further research.

O presente artigo procura indagar sobre as práticas e os limites da atuação dos movimentos sociais de luta por direito à moradia, tendo em vista novas e velhas reconfigurações dos territórios populares e o protagonismo das populações que neles habitam no âmbito dos conflitos urbanos e da formação dos sujeitos políticos. Através de relatos de campo, procura identificar alguns dos atores presentes no decorrer da luta pela regularização da ocupação, bem como seus entrelaçamentos, na disputa pela construção – ou não – da luta política. Dentro disso, a partir das cenas apreendidas no campo, o escrito levanta hipóteses a respeito dos caminhos traçados para a produção do espaço urbano, identificando estratégias que o formam e o caracterizam, bem como suas capacidades ou limites de emancipar os sujeitos em luta, tendo em vista as modulações da racionalidade estratégica neoliberal em solo brasileiro.

Ranaviruses are worldwide pathogens of ectothermic vertebrates that can threaten herptile conservation efforts. Identifying transmission routes is critical for understanding disease ecology and promoting species conservation. Frog virus 3 (FV3) DNA was detected in mosquitoes during a ranavirus outbreak in semicaptive box turtles, but the role that insect vectors play under natural conditions is unknown. To address this knowledge gap, we collected mosquito species known to take blood meals from reptiles and amphibians (Aedes canadensis, Culex erraticus, Culex territans, and Uranotaenia sapphirina) from mid-May to early August, 2014, at four study sites in Vermilion County, Illinois, two of which had historic or ongoing FV3 outbreaks in box turtles and amphibians. Mosquitoes were batched by date and species, DNA was extracted, and quantitative polymerase chain reaction was performed for detection of FV3. FV3 was not detected despite one of the sites having an active FV3 outbreak during the study period. Our findings indicate that FV3 detection is uncommon in mosquitoes during outbreak and nonoutbreak conditions at these sites in Illinois. Thus, we cannot establish that mosquitoes contribute to transmission during natural mortality events without performing further studies.

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method.

Phenylketonuria is an inborn error of metabolism caused by loss of function of the liver-expressed enzyme phenylalanine hydroxylase and is characterized by elevated systemic phenylalanine levels that are neurotoxic. Current therapies do not address the underlying genetic disease or restore the natural metabolic pathway resulting in the conversion of phenylalanine to tyrosine. A family of hepatotropic clade F adeno-associated viruses (AAVs) was isolated from human CD34+ hematopoietic stem cells (HSCs) and one (AAVHSC15) was utilized to deliver a vector to correct the phenylketonuria phenotype in Pahenu2 mice. The AAVHSC15 vector containing a codon-optimized form of the human phenylalanine hydroxylase cDNA was administered as a single intravenous dose to Pahenu2 mice maintained on a phenylalanine-containing normal chow diet. Optimization of the transgene resulted in a vector that produced a sustained reduction in serum phenylalanine and normalized tyrosine levels for the lifespan of Pahenu2 mice. Brain levels of phenylalanine and the downstream serotonin metabolite 5-hydroxyindoleacetic acid were restored. In addition, the coat color of treated mice darkened following treatment, indicating restoration of the phenylalanine metabolic pathway. Taken together, these data support the potential of an AAVHSC15-based gene therapy as an investigational therapeutic for phenylketonuria patients.

La presente investigación se realizó con el objetivo de determinar la relación que existe entre la gestión empresarial de las mypes de Pátzcuaro, Michoacán y el grado de innovación empresarial. Se realizó un trabajo de campo que incluyó la aplicación de un instrumento de recolección de datos diseñado en el trabajo de investigación 2021 de RELAYN “Caracterización de la cultura financiera de los micro y pequeños empresarios de Latinoamérica”. Los resultados obtenidos en una muestra del municipio de Pátzcuaro, indican que existe relación positiva entre las variables de estudio, y que es necesario ampliar los items utilizados para tener un panorama más amplio que permita profundizar en los aspectos de gestión e innovación empresarial. Abstract The objective of this research was determining the relationship that exists between entrepreneurial management in medium and small enterprises (SMEs) in Patzcuaro, Michoacan and the degree of innovation required to increase productivity and competitiveness in the market. This field work included the application of a data recollection instrument designed in research performed in 2021 by the Latin American Studies in Administration and Business Network (Red de Estudios Latinoamericanos en Administración y Negocios-RELAYN) “"Characterization of the financial culture of micro and small entrepreneurs in Latin America". The results obtained indicate a positive relationship between research variables and the need to expand the items in order to deepen managerial and business innovation aspects.

Purpose The presence of distinct child and adolescent mental health services (CAMHS) and adult mental health services (AMHS) impacts continuity of mental health treatment for young people. However, we do not know the extent of discontinuity of care in Europe nor the effects of discontinuity on the mental health of young people. Current research is limited, as the majority of existing studies are retrospective, based on small samples or used non-standardised information from medical records. The MILESTONE prospective cohort study aims to examine associations between service use, mental health and other outcomes over 24 months, using information from self, parent and clinician reports.Participants Seven hundred sixty-three young people from 39 CAMHS in 8 European countries, their parents and CAMHS clinicians who completed interviews and online questionnaires and were followed up for 2 years after reaching the upper age limit of the CAMHS they receive treatment at.Findings to date This cohort profile describes the baseline characteristics of the MILESTONE cohort. The mental health of young people reaching the upper age limit of their CAMHS varied greatly in type and severity: 32.8% of young people reported clinical levels of self-reported problems and 18.6% were rated to be ‘markedly ill’, ‘severely ill’ or ‘among the most extremely ill’ by their clinician. Fifty-seven per cent of young people reported psychotropic medication use in the previous half year.Future plans Analysis of longitudinal data from the MILESTONE cohort will be used to assess relationships between the demographic and clinical characteristics of young people reaching the upper age limit of their CAMHS and the type of care the young person uses over the next 2 years, such as whether the young person transitions to AMHS. At 2 years follow-up, the mental health outcomes of young people following different care pathways will be compared.Trial registration number NCT03013595.

Summary: The etiology of peripartum cardiomyopathy remains unknown. One hypothesis is that an increase in the 16-kDa form of prolactin is pathogenic and suggests that breastfeeding may worsen peripartum cardiomyopathy by increasing prolactin, while bromocriptine, which blocks prolactin release, may be therapeutic. An autoimmune etiology has also been proposed. The authors investigated the impact of breastfeeding on cellular immunity and myocardial recovery for women with peripartum cardiomyopathy in the IPAC (Investigations in Pregnancy Associated Cardiomyopathy) study. Women who breastfed had elevated prolactin, and prolactin levels correlated with elevations in CD8+ T cells. However, despite elevated prolactin and cytotoxic T cell subsets, myocardial recovery was not impaired in breastfeeding women. Key Words: breastfeeding, immune activation, peripartum cardiomyopathy

Baseline plasma electrophoresis profiles (EPH) are important components of overall health and may aid in the conservation and captive management of species. The aim of this study was to establish plasma protein fractions for free-ranging Blanding's turtles (Emydoidea blandingii) and evaluate differences due to age class (adult vs. sub-adult vs. juvenile), sex (male, female, or unknown), year (2018 vs. 2019), month (May vs. June vs. July), health status, and geographical location (managed vs. unmanaged sites). Blood samples were obtained from 156 Blanding's turtles in the summer of 2018 and 129 in 2019 at two adjacent sites in Illinois. Results of the multivariate analysis demonstrated that age class, sex, year, month, health status, and geographical location all contributed to the variation observed in free-ranging populations. Adult females had the highest concentration of many protein fractions, likely associated with reproductive activity. Juveniles had lower protein concentrations. Temperature and rainfall differences between years impacted concentrations between 2018 and 2019, while May and June of both years saw higher levels in some protein fractions likely due to peak breeding and nesting season. Individuals with evidence of trauma or disease also showed increased plasma protein fractions when compared to those that were considered healthy. The two sites showed a wide/large variation over the two years. All of these factors emphasize the importance of considering multiple demographic or environmental factors when interpreting the EPH fractions. Establishing ranges for these analytes will allow investigation into disease prevalence and other environmental factors impacting this endangered species.

BackgroundRabies is a viral disease of animals and people causing fatal encephalomyelitis if left untreated. Although effective pre- and post-exposure vaccines exist, they are not widely available in many endemic countries within Africa. Since many individuals in these countries remain at risk of infection, post-exposure healthcare-seeking behaviors are crucial in preventing infection and warrant examination.MethodologyA rabies knowledge, attitudes, and practices survey was conducted at 24 geographically diverse sites in Uganda during 2013 to capture information on knowledge concerning the disease, response to potential exposure events, and vaccination practices. Characteristics of the surveyed population and of the canine-bite victim sub-population were described. Post-exposure healthcare-seeking behaviors of canine-bite victims were examined and compared to the related healthcare-seeking attitudes of non-bite victim respondents. Wealth scores were calculated for each household, rabies knowledge was scored for each non-bitten survey respondent, and rabies exposure risk was scored for each bite victim. Logistic regression was used to determine the independent associations between different variables and healthcare-seeking behaviors among canine-bite victims as well as attitudes of non-bitten study respondents.ResultsA total of 798 households were interviewed, capturing 100 canine-bite victims and a bite incidence of 2.3 per 100 person-years. Over half of bite victims actively sought medical treatment (56%), though very few received rabies post-exposure prophylaxis (3%). Bite victims who did not know or report the closest location where PEP could be received were less likely to seek medical care (p = 0.05). Respondents who did not report having been bitten by a dog with higher knowledge scores were more likely to respond that they would both seek medical care (p = 0.00) and receive PEP (p = 0.06) after a potential rabies exposure event.ConclusionsThere was varying discordance between what respondents who did not report having been bitten by a dog said they would do if bitten by a dog when compared to the behaviors exhibited by canine-bite victims captured in the KAP survey. Bite victims seldom elected to wash their wound or receive PEP. Having lower rabies knowledge was a barrier to theoretically seeking care and receiving PEP among not bitten respondents, indicating a need for effective and robust educational programs in the country.

Objective Young people moving from child and adolescent mental health services (CAMHS) to adult mental health services (AMHS) are faced with significant challenges. To improve this state of affairs, there needs to be a recognition of the problem and initiatives and an urgent requirement for appropriate tools for measuring readiness and outcomes at the transfer boundary (16–18 years of age in Europe). The objective of this study was to develop and validate the Transition Readiness and Appropriateness Measure (TRAM) for assessing a young person’s readiness for transition, and their outcomes at the transfer boundary.Design MILESTONE prospective study.Setting Eight European Union (EU) countries participating in the EU-funded MILESTONE study.Participants The first phase (MILESTONE validation study) involved 100 adolescents (pre-transition), young adults (post-transition), parents/carers and both CAMHS and AMHS clinicians. The second phase (MILESTONE cohort study and nested cluster randomised trial) involved over 1000 young people.Results The development of the TRAM began with a literature review on transitioning and a review of important items regarding transition by a panel of 34 mental health experts. A list of 64 items of potential importance were identified, which together comprised the TRAM. The psychometric properties of the different versions of the TRAM were evaluated and showed that the TRAM had good reliability for all versions and low-to-moderate correlations when compared with other established instruments and a well-defined factor structure. The main results of the cohort study with the nested cluster randomised trial are not reported.Conclusion The TRAM is a reliable instrument for assessing transition readiness and appropriateness. It highlighted the barriers to a successful transition and informed clinicians, identifying areas which clinicians on both sides of the transfer boundary can work on to ease the transition for the young person.Trial registration number ISRCTN83240263 (Registered 23 July 2015), NCT03013595 (Registered 6 January 2017); Pre-results.

Based on extensive ethnographic fieldwork in Uzbekistan, this article looks at the way official state narratives are challenged by silent, unorganised, often unaware gestures of resistance at the bottom of a society. Footing on a framework suggested by Scott’s definition of infrapolitics (2012), we propose to incorporate informal practices in a definition of informality that is more inclusive and better explains the anatomy of a modern state, whose functioning rests on a combination of formal and informal practices. We suggest that this everyday dimension is of particular importance here when trying to understand the governance trajectories, as it allows to look critically, and from a broader perspective, at situations where individual and state perception of events, but also individual and state morality, diverge. By doing this, we propose that governance in transition states and societies may be regarded as a space where formal institutions and citizens (or informal institutions) compete for power and resources and thereby produce informal, alternative ‘legal orders’ and mechanisms that regulate public life in a given area. We will suggest that such a space of informal negotiation is vital in contexts where collective mobilisation and public articulation of social claims is not a preferred, or even available, strategy for citizens.

Many applications have been developed for electronic data collection. However, offline field navigation tools incorporating secure electronic data capture and field visit tracking are currently scarce. We created an R-Shiny application, HTrack (Household Tracking), for use on encrypted Android devices in the field. The application was implemented in the Communities Organized to Prevent Arboviruses (COPA) project, a study beginning in 2018 to better understand arboviral disease incidence in 38 communities in Puerto Rico. The application was used to navigate to randomly selected structures and capture visit outcomes after conducting multiple visits for participant recruitment. It also served as a bridge to an alternate software, Epi Info, to collect participant-level questionnaire data. This application successfully captured each visit outcome and improved the logistics of field level activities for the COPA project, eliminating the use of paper maps for navigation. We show the development of HTrack and comment on the limitations and strengths of this application and further improvements.

Targeted gene integration via precise homologous recombination (HR)-based gene editing has the potential to correct genetic diseases. AAV (adeno-associated virus) can mediate nuclease-free gene integration at a disease-causing locus. Therapeutic application of AAV gene integration requires quantitative molecular characterization of the edited sequence that overcome technical obstacles such as excess episomal vector genomes and lengthy homology arms. Here we describe a novel molecular methodology that utilizes quantitative next-generation sequencing to characterize AAV-mediated targeted insertion and detects the presence of unintended mutations. The methods described here quantify targeted insertion and query the entirety of the target locus for the presence of insertions, deletions, single nucleotide variants (SNVs) and integration of viral components such as inverted terminal repeats (ITR). Using a humanized liver murine model, we demonstrate that hematopoietic stem-cell derived AAVHSC15 mediates in vivo targeted gene integration into human chromosome 12 at the PAH (phenylalanine hydroxylase) locus at 6% frequency, with no sign of co-incident random mutations at or above a lower limit of detection of 0.5% and no ITR sequences at the integration sites. Furthermore, analysis of heterozygous variants across the targeted locus using the methods described shows a pattern of strand cross-over, supportive of an HR mechanism of gene integration with similar efficiencies across two different haplotypes. Rapid advances in the application of AAV-mediated nuclease-free target integration, or gene editing, as a new therapeutic modality requires precise understanding of the efficiency and the nature of the changes being introduced to the target genome at the molecular level. This work provides a framework to be applied to homologous recombination gene editing platforms for assessment of introduced and natural sequence variation across a target site.

The acute phase response is a highly conserved reaction to infection, inflammation, trauma, stress, and neoplasia. Acute phase assays are useful for wildlife health assessment, however, they are infrequently utilized in reptiles. This study evaluated erythrocyte sedimentation rate (ESR) in eastern (Terrapene carolina carolina) and ornate box turtles (Terrapene ornata ornata) and hemoglobin-binding protein (HBP) in T. ornata. Erythrocyte sedimentation rate in 90 T. carolina and 105 T. ornata was negatively associated with packed cell volume and was greater in unhealthy turtles (p < 0.05). Female T. ornata had higher ESR values than males (p < 0.05). Measurement of ESR with a microhematocrit tube proportionally overestimated values from a commercial kit (Winpette), though both methods may retain utility with separate reference intervals. Hemoglobin-binding protein concentration in 184 T. ornata was significantly increased in adults and unhealthy turtles (p < 0.05). Erythrocyte sedimentation rate values were similar between seasons and populations, and HBP values were consistent between years, indicating that these analytes may have more stable baseline values than traditional health metrics in reptiles. This study demonstrates that ESR and HBP are promising diagnostics for health assessment in wild box turtles. Incorporating these tests into wild herptile health assessment protocols may support conservation efforts and improve ecosystem health monitoring.

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.