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Abstract Background The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored. Results By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells. Conclusions Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

IntroductionPreventing potential foreign animal diseases is a high priority, with re-emerging threats such as African Swine Fever emerging close to North American borders. The Secure Pork Supply (SPS) plan provides a voluntary framework for swine producer biosecurity planning and disease outbreak preparedness. However, biosecurity knowledge varies greatly among swine veterinarians, managers, and caretakers within the industry, which impacts the understanding, quality, implementation and biosecurity plan agreements with the SPS guidelines unless review procedures and quality control mechanisms are in place. Therefore, this study aimed to describe and identify the level of biosecurity planning agreements between producer-and/or swine veterinarian-made biosecurity plans for commercial swine sites and the SPS plan guidelines during a review process.Material and methodsBiosecurity maps (N = 368) and written plans (N = 247) were obtained from six Midwest swine companies/veterinary clinics. Maps were evaluated on accuracy and placement of mandatory map features based on SPS guidelines, and discrepancies between the development of producer-made biosecurity maps and written biosecurity plans. Multivariable mixed logistic regression analyses were conducted to identify differences in SPS planning accuracy based on herd size, production stage, and characteristics related to geographical site location (e.g., land cover type and expected feral swine population density in the region).ResultsIn this study, 55.8% (205/368) of all provided biosecurity maps had to be revised due to misplaced or missing map features. In addition, 80.9% (200/247) of the written plans had one or more conflicts with the corresponding biosecurity maps. The main biosecurity planning issues involved feed delivery activities, where the mapping of vehicle movements (89.9%, 222/247) were in direct conflict with the written SPS plans. Sites located in areas with a moderate expected feral swine population density had 3-fold increased odds of needing map revisions compared to sites with low expected feral swine population density. Sites located in predominately farmland had 7.3% lower odds of having biosecurity map and SPS plan conflicts for every 1.0% increase in farmland landcover in a 10-km radius around the swine site.DiscussionHuman oversight or lack of knowledge regarding biosecurity planning and implementation is common, which may culminate in important preparedness shortcomings in disease prevention and control strategies for U.S. swine farms. Future efforts should focus on additional biosecurity training for swine producers and veterinarians alongside with quality control benchmarking of producer made plans.

The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.

Abstract The United States’ swine industry is under constant threat of foreign animal diseases, which may emerge without warning due to the globalized transportation networks moving people, animals, and products. Therefore, having disease control and elimination protocols in place prior to pathogen introduction is paramount for business continuity and economic recovery. During extraordinary circumstances, it may become necessary to depopulate large populations of animals, including swine, as a disease containment measure. Currently approved depopulation methods for swine present significant logistical challenges when scaled to large populations or performed in field conditions. In the United States, water-based foam is currently approved for poultry depopulation, and recent field studies demonstrate water-based foam is an effective depopulation alternative for swine. While effective, the speed at which water-based foam induces loss of consciousness prior to death, a major welfare consideration, has not been adequately investigated. In this study, 12 nursery pigs were terminated using water-based medium-expansion foam to quantify the time to induce loss of consciousness and ultimately brain death. Each pig was implanted with subdermal electrodes to capture electroencephalographic data, placed in a body sling, and suspended in a plastic bulk container that was subsequently filled with water-based foam. Electroencephalographic data was recorded for 15 min, during which the pigs remained immersed in the water-based foam. Conservatively, average (± SD) time to unconsciousness and brain death was 1 min, 53 s ± 36 s and 3 min, 3 s ± 56 s, respectively. The relatively rapid loss of consciousness compared to other methods limits the amount of distress and is overall a positive finding for the welfare of the pigs that might be depopulated with water-based foam. The findings of this study add additional evidence supporting the use of water-based medium-expansion foam for an emergency depopulation of swine.

Abstract Background In commercial pig farming, sick or injured sows are often treated by producers or hired staff. To date, limited quantitative data exists on treatment compliance and the possible effect on sow longevity post-treatment. The objective of the study was to quantify on-farm compliance of treatment selection, frequency, and dosage, as well as to investigate the association between body condition scores (BCS) and other sow-level factors on post-treatment cull risk. Results On-farm treatment records, including culling reason or reason of death up to 6 months post-treatment, production records and sow characteristics were obtained for 134 sows over an 8-week period. Treatment compliance was based on the accuracy of recorded treatments compared to the herd veterinarian’s established treatment guidelines. Univariable and multivariable logistic regression models including treatment reason, treatment compliance, BCS, parity, production stage and production metrics, were constructed to investigate associations between those variables and sow culling or death. This study found low compliance for on-farm sow treatment protocols, with only 22.4% (30/134) of the sows receiving correct and complete treatment during the duration of the study. No effect of individual treatment components (drug, dosage, or frequency) on sow culling was observed. A trend for an interaction between treatment compliance and BCS was found, and parity and number of piglets born alive were identified as predictors for sow maintenance in the herd. Conclusions On-farm sow treatment compliance was low, resulting in that approximately 80% of the enrolled sows were not treated according to existing guidelines. Non-compliance of treatment guidelines did not seem to affect the risk of culling in treated sows but may have prolonged any associated pain, recovery time and negatively impacted the sow welfare during that time period.

Polycomb Repressive Complex 2, an important regulator of embryonic development, exists in two configurations, PRC2.1 and PRC2.2. Here the authors dissect the functional contributions of these two PRC2 subunits and observed complex-specific alterations in the cell state of pluripotent and early differentiating cells.

Respiratory infections caused by Ornithobacterium rhinotrachealis (ORT) and Pasteurella multocida (PM) bacteria are significant threats to the poultry industry by causing economic losses and welfare issues. Due to characterization difficulties and underutilization of epidemiological tools, description of the spatio-temporal spread of these diseases in the field is limited. The objectives of this retrospective observational cross-sectional study were to (a) investigate the existence of space–time clusters (hotspots); and (b) investigate the association between genetic similarity and spatial proximity for both pathogens using molecular typing and a recently developed Core-Genome Multilocus Sequencing Typing (cgMLST) scheme. ORT (n = 103) and PM (n = 69) isolates from confirmed disease outbreaks from one commercial company between 2013 and 2021 were obtained from a veterinary diagnostic laboratory, characterized using a cgMLST scheme and visualized using a minimum spanning tree. Spatio-temporal cluster analysis using SaTScanTM and a Spearman’s rank correlation were performed to investigate clustering and any association between allelic diversity and geospatial distance. The cgMLST sequencing revealed three allelic clusters for ORT and thirteen clusters for PM. The spatio-temporal analysis revealed two significant clusters for PM, one with a 259.3 km cluster containing six cases between May and July 2018 and a 9 km cluster containing five cases between February 2019 and February 2021. No spatio-temporal clusters were found for ORT. A weak negative correlation between allelic diversity and geospatial distance was observed for ORT (r = −0.04, p < 0.01) and a weak positive correlation was observed for PM (r = 0.11, p < 0.01). This study revealed regional spatio-temporal clusters for PM in commercial turkey sites between 2018 and 2021 and provided additional insight into bacterial strain subgroups and the geographical spread of ORT and PM over time.

The detection and co-circulation of multiple variants of porcine reproductive and respiratory syndrome virus (PRRSV) have been observed and reported in swine. However, the potential long-term impact of multiple prevailing PRRSV variants on pig-performance is not yet fully understood. The primary objective of this study was to describe the genetic variation of PRRSV in processing fluid (PF), oral fluid (OF), and tonsil scraping (TS) specimens from five swine farms with different production types and PRRS status over a period of time (~1 year). Furthermore, the association between PRRSV prevalence and production parameters was investigated. Results showed that PRRSV was detected by RT-qPCR in 21–25% of all types of specimens. In breeding farms, PRRSV detection in PF and/or TS samples was correlated with stillborn and mummified fetuses, and pre-weaning mortality throughout the study period. Although ORF5 sequences were obtained in

Abstract Background Adrenal hypoplasia congenita (AHC) is an X-linked disorder that affects the adrenal cortex and hypothalamus-pituitary-gonadal axis (HPG), leading to primary adrenocortical insufficiency (PAI) and hypogonadotropic hypogonadism. AHC is caused by a mutation in the DAX-1 gene (NR0B1). More commonly, this disease is characterized by early-onset PAI, with symptoms in the first months of life. However, a less severe phenotype termed late-onset AHC has been described, as PAI signs and symptoms may begin in adolescence and adulthood. Here we describe a family report of a novel mutation within NR0B1 gene and variable reproductive phenotypes, including spontaneous fertility, in a very late-onset X-linked AHC kindred. Case presentation Three affected maternal male relatives had confirmed PAI diagnosis between 30 y and at late 64 y. The X-linked pattern has made the endocrinology team to AHC suspicion. Regarding the HPG axis, all males presented a distinct degree of testosterone deficiency and fertility phenotypes, varying from a variable degree of hypogonadism, oligoasthenoteratozoospermia to spontaneous fertility. Interestingly, the other five maternal male relatives unexpectedly died during early adulthood, most likely due to undiagnosed PAI/adrenal crisis as the probable cause of their premature deaths. Sequencing analysis of the NR0B1 gene has shown a novel NR0B1 mutation (p.Tyr378Cys) that segregated in three AHC family members. Conclusions NR0B1 p.Tyr378Cys segregates in an AHC family with a variable degree of adrenal and gonadal phenotypes, and its hemizygous trait explains the disease in affected family members. We recommend that NR0B1 mutation carriers, even those that are allegedly asymptomatic, be carefully monitored while reinforcing education to prevent PAI and consider early sperm banking when spermatogenesis still viable.

Objective: Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK that bind to the allosteric drug and metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. This study aimed to illuminate the mechanisms by which ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e., ADaM-site binding small molecules and the prodrug AICAR. Methods: The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2 as well as whole-body AMPKγ3-deficient mouse models. In vitro complex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by western blotting in muscle lysate. To investigate the transferability of these studies, we treated diet-induced obese mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle. Results: Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2β2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2β2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscles. In vivo PF739 treatment lowered blood glucose levels and increased muscle AMPKγ1-complex activity 2-fold, while AMPKα2β2γ3 activity was not affected. Conclusions: ADaM-site binding AMPK activators increase glucose uptake independently of AMPKγ3. Co-incubation with PF739 or 991 and AICAR potentiates the effects on muscle glucose uptake and AMPK activation. In vivo, PF739 lowers blood glucose and selectively activates muscle AMPKγ1-complexes. Collectively, this suggests that pharmacological activation of AMPKγ1-containing complexes in skeletal muscle can increase glucose uptake and can lead to blood glucose lowering.

Klinefelter syndrome (KS) displays a broad dysmorphological, endocrinological, and neuropsychological clinical spectrum. We hypothesized that the neurocognitive dysfunction present in KS relies on an imbalance in X-chromosome gene expression. Thus, the X-chromosome inactivation (XCI) pattern and neurocognitive X-linked gene expression were tested and correlated with intelligence quotient (IQ) scores. We evaluated 11 KS patients by (a) IQ assessment, (b) analyzing the XCI patterns using both HUMARA and ZDHHC15 gene assays, and (c) blood RT-qPCR to investigate seven X-linked genes related to neurocognitive development (GTPBP6, EIF2S3, ITM2A, HUWE1, KDM5C, GDI1, and VAMP7) and XIST in comparison with 14 (male and female) controls. Considering IQ 80 as the standard minimum reference, we verified that the variability in IQ scores in KS patients seemed to be associated with the XCI pattern. Seven individuals in the KS group presented a random X-inactivation (RXI) and lower average IQ than the four individuals who presented a skewed X-inactivation (SXI) pattern. The evaluation of gene expression showed higher GTPBP6 expression in KS patients with RXI than in controls (p = 0.0059). Interestingly, the expression of GTPBP6 in KS patients with SXI did not differ from that observed in controls. Therefore, our data suggest for the first time that GTPBP6 expression is negatively associated with full-scale IQ under the regulation of the type of XCI pattern. The SXI pattern may regulate GTPBP6 expression, thereby dampening the impairment in cognitive performance and playing a role in intelligence variability in individuals with KS, which warrants further mechanistic investigations.

Abstract Background Different pathogenic germline mutations in the RET oncogene are identified in MEN 2, a hereditary syndrome characterized by medullary thyroid carcinoma (MTC) and other endocrine tumors. Although genetic predisposition is recognized, not all RET mutation carriers will develop the disease during their lifetime or, likewise, RET mutation carriers belonging to the same family may present clinical heterogeneity. It has been suggested that a single germline mutation might not be sufficient for development of MEN 2-associated tumors and a somatic bi-allelic alteration might be required. Here we investigated the presence of somatic second hit mutation in the RET gene in MTC. Methods We integrated Multiplex Ligation-dependent Probe Amplification (MLPA) and whole exome sequencing (WES) to search for copy number alteration (CNA) in the RET gene in MTC samples and medullary thyroid cell lines (TT and MZ-CR-1). We next found reads spanning exon-exon boundaries on RET, an indicative of retrocopy. We subsequently searched for RET retrocopies in the human reference genome (GRCh37) and in the 1000 Genomes Project data, by looking for reads reporting joined exons in the RET locus or distinct genomic regions. To determine RET retrocopy specificity and recurrence, DNA isolated from sporadic and MEN 2-associated MTC (n = 37), peripheral blood (n = 3) and papillary thyroid carcinomas with RET fusion (n = 10) samples were tested using PCR-sequencing methodology. Results Through MLPA we have found evidence of CNA in the RET gene in MTC samples and MTC cell lines. WES analysis reinforced the presence of the CNA and hinted for a retroposed copy of RET not found in the human reference genome and 1.000 Genomes Project. Extended analysis confirmed the presence of a somatic MTC-related retrocopy of RET in both sporadic and hereditary tumors. We further unveiled a recurrent (28%) novel point mutation (p.G548 V) found exclusively in the retrocopy of RET. The mutation was also found in cDNA of mutated samples, suggesting it might be functional. Conclusion We here report a somatic specific RET retroposed copy in MTC samples and cell lines. Our results support the idea that generation of retrocopies in somatic cells is likely to contribute to MTC genesis and progression.

The threat of foreign animal disease introduction through contaminated animal products, feed ingredients, and wildlife vectors have highlighted the need for additional approved methods for mass depopulation of swine under emergency scenarios, especially methods that can be applied to pigs across all production phases. The market disruption within the swine industry due to the SARS-CoV-2 pandemic has demonstrated this lack of preparation. The objective of this study was to validate water-based foam as a mass depopulation method for suckling (18 to 24 days of age) and finisher stage (63 to 100 days of age) pigs. Finisher pigs (n = 31, originally 32 but one finisher pig died prior to foaming), allocated as 9 triads and 1 set of 4 pigs, in 10 total replicates, and suckling pigs (n = 32), randomly allocated to two replicates, were completely covered in water-based medium-expansion foam for a 15-min dwell time in a bulk container. Container fill time for the trials were 6.5 ± 0.68 s and 5.3 ± 0.03 s for finisher and suckling pig replicates, respectively. Average (± SD) time for cessation of movement was 105 ± 39.1 s (s) for finisher pigs and 79.5 ± 10.5 s for suckling pigs. After completion of the 15-min dwell time in the foam, all pigs were confirmed dead upon removal from the container. The results from the present study suggest that the use of water-based foam can be an effective means of mass depopulation for suckling and finisher stage pigs, supporting previous research on the application to adult swine.

Body condition scoring (BCS) is a traditional visual technique often using a five-point scale to non-invasively assess fat reserves in cattle. However, recent studies have highlighted the potential in automating body condition scoring using imaging technology. Therefore, the objective was to implement a commercially available automated body condition scoring (ABCS) camera system to collect data for developing a predictive equation of body condition dynamics throughout the lactation period. Holstein cows (n = 2343, parity = 2.1 ± 1.1, calving BCS = 3.42 ± 0.24), up to 300 days in milk (DIM), were scored daily using two ABCS cameras mounted on sort-gates at the milk parlor exits. Scores were reported on a 1 to 5 scale in 0.1 increments. Lactation number, DIM, disease status, and 305d-predicted-milk-yield (305PMY) were used to create a multivariate prediction model for body condition scores throughout lactation. The equation derived from the model was: ABCSijk = 1.4838 − 0.00452 × DIMi − 0.03851 × Lactation numberj + 0.5970 × Calving ABCSk + 0.02998 × Disease Status(neg)l − 1.52 × 10−6 × 305PMYm + eijklm. We identified factors which are significant for predicting the BCS curve during lactation. These could be used to monitor deviations or benchmark ABCS in lactating dairy cows. The advantage of BCS automation is that it may provide objective, frequent, and accurate BCS with a higher degree of sensitivity compared with more sporadic and subjective manual BCS. Applying ABCS technology in future studies on commercial dairies may assist in providing improved dairy management protocols based on more available BCS.

Objective: Evidence for AMP-activated protein kinase (AMPK)-mediated regulation of skeletal muscle metabolism during exercise is mainly based on transgenic mouse models with chronic (lifelong) disruption of AMPK function. Findings based on such models are potentially biased by secondary effects related to a chronic lack of AMPK function. To study the direct effect(s) of AMPK on muscle metabolism during exercise, we generated a new mouse model with inducible muscle-specific deletion of AMPKα catalytic subunits in adult mice. Methods: Tamoxifen-inducible and muscle-specific AMPKα1/α2 double KO mice (AMPKα imdKO) were generated by using the Cre/loxP system, with the Cre under the control of the human skeletal muscle actin (HSA) promoter. Results: During treadmill running at the same relative exercise intensity, AMPKα imdKO mice showed greater depletion of muscle ATP, which was associated with accumulation of the deamination product IMP. Muscle-specific deletion of AMPKα in adult mice promptly reduced maximal running speed and muscle glycogen content and was associated with reduced expression of UGP2, a key component of the glycogen synthesis pathway. Muscle mitochondrial respiration, whole-body substrate utilization, and muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion of AMPKα subunits in adult mice. Conclusions: Inducible deletion of AMPKα subunits in adult mice reveals that AMPK is required for maintaining muscle ATP levels and nucleotide balance during exercise but is dispensable for regulating muscle glucose uptake, FA oxidation, and substrate utilization during exercise.

The U.S. swine industry is currently inadequately prepared to counteract the increasing threat of high-consequence diseases. Although approved and preferred depopulation guidelines exist, ventilation shutdown (VSD+) is currently the only method being deployed during a state of emergency to depopulate large swine populations. However, the permitted use of VSD+ during constrained circumstances has been criticized due to raised swine welfare concerns. The objective of this study was to investigate the effectiveness of carbon dioxide gas (CO2), nitrogen gas (N2), compressed air foam (CAF), compressed nitrogen foam (CAF-N2) and aspirated foam (AF) during a 15-min dwell time on adult swine in an emergency depopulation situation. A small-scale trial using 12 sows per depopulation method showed the highest efficiency to induce cessation of movement for AF and CO2 (186.0 ± 48 vs. 202.0 ± 41, s ± SD). The ease of implementation and safety favored AF for further investigation. A large-scale field study using AF to depopulate 134 sows in modified rendering trailers showed a mean fill time of 103.8 s (SD: 5.0 s) and cessation of movement of 128.0 s (SD: 18.6 s) post filling. All sows were confirmed dead post-treatment for both trials. The implementation of AF in modified rendering trailers may allow for a safe and reliable method that allows for the expedient and mobile depopulation of both small and large numbers of sows during an emergency.

ABSTRACT Measuring thyroid hormones is an important aspect for the study of metabolism and for monitoring diseases in both human and animal models. The traditional method for hormone measurement in rats is the radioimmunoassay (RIA). However, the RIA is associated with some practical disadvantages, including the use of radioactive material, the need for specialized equipment and expert staff, the short shelf-life of kits according to the half-life of the radioisotope and high costs. The objective of this study was to develop a new cost-effective method for measuring TSH levels in rats that avoids the use of radioactive material. We developed an in-house competitive immunoassay using a reference standard, polyclonal antibody produced in rabbits and biotinylated antigen. This method was tested in 64 Wistar rats that were divided into a control group (n = 41) and a group with hypothyroidism (n = 23). Our assay demonstrated an analytical sensitivity of 0.24 ng/mL (n = 12) and an intra-assay coefficient of variation (CV) of 8.9% for sera with TSH levels of 1.5 ng/mL and 13.2% for sera with TSH levels of 17.5 ng/mL (n = 14). The inter-assay CV was 13.5% for sera with TSH levels of 1.4 ng/mL and 14.5% for TSH levels of 18.2 ng/mL (n = 5). The analysis of mean TSH levels in control rats (5.06 ± 0.5701) and hypothyroid rats (51.09 ± 5.136) revealed a statistically significant difference (p < 0.001) between the groups. This method showed good sensitivity, can be automated and is low-cost compared with RIA. Our method offers a viable alternative for TSH measurement in rats.

An approach is demonstrated to visualise overhead line failure rates and estimated wind power output during extreme wind events on transmission networks. Reanalysis data is combined with network data and line failure models to illustrate spatially resolved line failure probability with data corrected for asset altitude and exposure. Wind output is estimated using a corrected power curve to account for high speed shutdown with wind speed corrected for altitude. Case studies demonstrate these methods' application on representations of real networks of different scales. The proposed methods allow users to determine at-risk regions of overhead line networks and to estimate the impact on wind power output. Such techniques could equally be applied to forecasted weather conditions to aid in resilience planning. The methods are shown to be particularly sensitive to the weather data used, especially when modelling risk on overhead lines, but are still shown to be useful as an indicative representation of system risk. The techniques also provide a more robust method of representing weather-related failure rates on lines considerate of the altitude, voltage level, and their varying exposure to weather conditions than current techniques typically provide, which can be used to usefully represent failure probability of lines during storms.

ABSTRACT Objective To verify the presence of variants in HNF1B in a sample of the Brazilian population selected according to the presence of renal cysts associated with hyperglycemia. Subjects and methods We evaluated 28 unrelated patients with clinical suspicion of HNF1B mutation because of the concomitant presence of diabetes mellitus (DM) or prediabetes and renal cysts. Genotyping was accomplished using Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). In positive cases, available relatives were recruited. Results We found two patients with HNF1B mutations. The first presented the variant p.Pro328Leufs*48(c.983delC) and had DM, renal cysts, and hypomagnesemia. The second presented a heterozygous whole gene deletion in HNF1B, DM, renal cysts, body and tail pancreatic agenesis, and hypomagnesemia; this alteration was also found in his two siblings and his father. Conclusion The recruitment of suspected cases of HNF1B gene mutations in Brazilians due to hyperglycemia and renal cysts presents two positive cases. Our cases contribute to the annotation of clinical and biochemical phenotypes of this rare form of maturity-onset diabetes of the young (MODY).

We performed nuclear magnetic resonance measurements on lead(II) nitrate Pb(NO_{3})_{2} in aqueous solution and on the hexafluoridoplumbate(IV) [PbF_{6}]^{2−} ion in acetonitrile. Combined with new relativistic coupled cluster and relativistic density functional theory calculations of the shielding constant, we obtained a magnetic moment of μ(^{207}Pb)=0.59102(18)μ_{N} that is in clear disagreement with the tabulated value of +0.592583(9)μ_{N}. Similarly as in the case of ^{209}Bi this might be caused by an underestimated chemical shift in the aqueous solution of the nitrate. The consequences for a test of QED in strong magnetic fields by laser spectroscopy of the hyperfine splitting in Pb^{81+} and for the magnetic moments of short-lived lead isotopes are discussed.

Objective Consuming a low-iodine diet (LID) is a widely accepted practice before administering radioiodine (131I) to evaluate and to treat thyroid disease. Although this procedure is well established for the management of patients with differentiated thyroid cancer, its use in patients with benign disease is unclear. So, we aimed to evaluate the influence of a LID on the outcome in patients with Graves’ disease (GD) treated with131I. Subjects and methods We evaluated 67 patients with GD who were divided into 2 groups: one group (n = 31) consumed a LID for 1-2 weeks, and the second group (n = 36) was instructed to maintain a regular diet (RD). Results The LID group experienced a 23% decrease in urinary iodine after 1 week on the diet and a significant 42% decrease after 2 weeks on the diet. The majority (53%) of the patients in the LID group had urinary iodine levels that were consistent with deficient iodine intake. However, there was no difference in the rate of hyperthyroidism’s cure between the LID and the RD groups 6 months after 131I therapy. Furthermore, the therapeutic efficacy did not differ in patients with varying degrees of sufficient iodine intake (corresponding urinary iodine levels: < 10 μg/dL is deficient; 10-29.9 μg/dL is sufficient; and > 30 μg/dL is excessive). Conclusion In the present study, we demonstrated that although a LID decreased urinary iodine levels, those levels corresponding with sufficient or a mild excess in iodine intake did not compromise the therapeutic efficacy of131I for the treatment of GD.

Neck ventroflexion in cats has different causes; however, the most common is the hypokalemia associated with flaccid paralysis secondary to chronic renal failure. In humans, the most common causes of acute flaccid paralysis are hypokalemia precipitated by thyrotoxicosis and familial forms linked to mutations in sodium, potassium, and calcium channel genes. Here, we describe the sequencing and analysis of skeletal muscle ion channels in Felis catus that could be related to periodic paralyses in humans, contributing to the understanding of the genetic susceptibility to feline neck ventroflexion and paralysis. We studied genomic DNA from eleven cats, including five animals that were hyperthyroid with hypokalemia, although only one presented with muscle weakness, and six healthy control domestic cats. We identified the ion channel ortholog genes KCNJ2, KCNJ12, KCNJ14, CACNA1S and SCN4A in the Felis catus genome, together with several polymorphic variants. Upon comparative alignment with other genomes, we found that Felis catus provides evidence for a high genomic conservation of ion channel sequences. Although we hypothesized that neck ventroflexion in cats could be associated with a thyrotoxic or familial periodic paralysis channel mutation, we did not identify any previously detected human channel mutation in the hyperthyroid cat presenting hypokalemia. However, based on the small number of affected cats in this study, we cannot yet rule out this molecular mechanism. Notwithstanding, hyperthyroidism should still be considered as a differential diagnosis in hypokalemic feline paralysis.

Body condition scoring (BCS) is the management practice of assessing body reserves of individual animals by visual or tactile estimation of subcutaneous fat and muscle. Both high and low BCS can negatively impact milk production, disease, and reproduction. Visual or tactile estimation of subcutaneous fat reserves in dairy cattle relies on their body shape or thickness of fat layers and muscle on key areas of the body. Although manual BCS has proven beneficial, consistent qualitative scoring can be difficult to implement. The desirable BCS range for dairy cows varies within lactation and should be monitored at multiple time points throughout lactation for the most impact, a practice that can be hard to implement. However, a commercial automatic BCS camera is currently available for dairy cattle (DeLaval Body Condition Scoring, BCS DeLaval International AB, Tumba, Sweden). The objective of this study was to validate the implementation of an automated BCS system in a commercial setting and compare agreement of the automated body condition scores with conventional manual scoring. The study was conducted on a commercial farm in Indiana, USA, in April 2017. Three trained staff members scored 343 cows manually using a 1 to 5 BCS scale, with 0.25 increments. Pearson’s correlations (0.85, scorer 1 vs. 2; 0.87, scorer 2 vs. 3; and 0.86, scorer 1 vs. 3) and Cohen’s Kappa coefficients (0.62, scorer 1 vs. 2; 0.66, scorer 2 vs. 3; and 0.66, scorer 1 vs. 3) were calculated to assess interobserver reliability, with the correlations being 0.85, 0.87, and 0.86. The automated camera BCS scores were compared with the averaged manual scores. The mean BCS were 3.39 ± 0.32 and 3.27 ± 0.27 (mean ± SD) for manual and automatic camera scores, respectively. We found that the automated body condition scoring technology was strongly correlated with the manual scores, with a correlation of 0.78. The automated BCS camera system accuracy was equivalent to manual scoring, with a mean error of −0.1 BCS and within the acceptable manual error threshold of 0.25 BCS between BCS (3.00 to 3.75) but was less accurate for cows with high (>3.75) or low (

Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(−)β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1), Pinus taeda α-pinene synthase [PtTPS-(+)αpin], and P. taeda α-farnesene synthase (PtαFS) were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers.

Two barium hexafluoridoosmates, Ba(OsF6)2 and BaOsF6, were synthesized and were characterized for the first time using X-ray powder and single crystal diffraction, IR spectroscopy, as well as NMR spectroscopy in anhydrous hydrogen fluoride. Ba(OsF6)2 crystallizes in the space group type P21/c with the cell parameters a = 6.4599(4), b = 10.7931(8), c = 14.7476(10) Å, β = 115.195(5)°, V = 930.42(12) Å3, Z = 4 at 293 K. BaOsF6 crystallizes in the space group type R 3 ¯ with the cell parameters a = 7.3286(10), c = 7.2658(15) Å, V = 337.95(12) Å3, Z = 3 at 100 K. Additionally, we have obtained the compounds Ba(OsF6)2∙3BrF3, Ba(OsF6)2∙HF, Ba(OsF6)2∙6H2O from the respective solvents, and Ba(OsF6)2.

Acute leukemia following treatment with Iodine131 is a rare event. The possible carcinogenic effect of Iodine131 is still not clear and a large series of cases did not show an increased incidence of cancer. A case of AML t(8;21), three years after Iodine131 treatment for hyperthyroidism, is reported. Secondary AML with t(8;21) is described following exposure to drugs that target topoisomerase II and radiotherapy. The controversial potential of Iodine131 as a leukemogenic agent and the fact that t(8;21) is also found in de-novo AML, emphasize the problem in establishing a relationship between these events although this potential can not be ruled out.

The beryllium amide and imide complexes [Be(HNMes)2]3, [(py)2Be(HNMes)2], [Be(HNDipp)2]2, [Be(NPh2)(μ2-HNDipp)]2 and [Be(NCPh2)2]3 have been prepared and characterised with NMR and IR spectroscopy as well as single crystal X-ray diffraction. Analysis of the localised molecular orbitals (LMOs) and intrinsic atomic orbital (IAO) atomic charges in the framework of the intrinsic bond orbital (IBO) localization method revealed a covalent bonding network consisting of 2-electron–2-centre and 2-electron–3-centre σ bonds, in which one electron pair of the anionic N-donor ligands is involved. The electron deficiency at the beryllium atoms is partially compensated through additional electron donation from the lone pair at the nitrogen atoms. [ABSTRACT FROM AUTHOR]

Attempts to create a novel Mg−Be bond by reaction of [(DIPePBDI*)MgNa]2 with Be[N(SiMe3)2]2 failed; DIPePBDI*=HC[(tBu)C=N(DIPeP)]2, DIPeP=2,6‐Et2C‐phenyl. Even at elevated temperatures, no conversion was observed. This is likely caused by strong steric shielding of the Be center. A similar reaction with the more open Cp*BeCl gave in quantitative yield (DIPePBDI*)MgBeCp* (1). The crystal structure shows a Mg−Be bond of 2.469(4) Å. Homolytic cleavage of the Mg−Be bond requires ΔH=69.6 kcal mol−1 (cf. CpBe−BeCp 69.0 kcal mol−1 and (DIPPBDI)Mg−Mg(DIPPBDI) 55.8 kcal mol−1). Natural‐Population‐Analysis (NPA) shows fragment charges: (DIPePBDI*)Mg +0.27/BeCp* −0.27. The very low NPA charge on Be (+0.62) compared to Mg (+1.21) and the strongly upfield 9Be NMR signal at −23.7 ppm are in line with considerable electron density on Be and the formal oxidation state assignment of MgII−Be0. Despite this Mgδ+−Beδ− polarity, 1 is extremely thermally stable and unreactive towards H2, CO, N2, cyclohexene and carbodiimide. It reacted with benzophenone, azobenzene, phenyl acetylene, CO2 and CS2. Reaction with 1‐adamantyl azide led to reductive coupling and formation of an N6‐chain. The azide reagent also inserted in the Cp*−Be bond. The inertness of 1 is likely due to bulky ligands protecting the Mg−Be unit. [ABSTRACT FROM AUTHOR]

ABSTRACT Objective: Initial diagnosis of medullary thyroid carcinoma (MTC) is frequently associated with advanced stages and a poor prognosis. Thus, the need for earlier diagnoses and detection in relatives at risk for the disease has led to increased use of RET genetic screening. Subjects and methods: We performed RET screening in 247 subjects who were referred to the Brazilian Research Consortium for Multiple Endocrine Neoplasia (BRASMEN) Center in the State of Ceará. Direct genetic sequencing was used to analyze exons 8, 10, 11, and 13-16 in MTC index cases and specific exons in at risk relatives. Afterward, clinical follow-up was offered to all the patients with MTC and their affected relatives. Results: RET screening was performed in 60 MTC index patients and 187 at-risk family members. At the initial clinical assessment of the index patients, 54 (90%) were diagnosed with apparently sporadic disease and 6 (10%) diagnosed with hereditary disease. After RET screening, we found that 31 (52%) index patients had sporadic disease, and 29 (48%) had hereditary disease. Regarding at-risk relatives, 73/187 were mutation carriers. Mutations in RET codon 804 and the rare p.M918V mutation were the most prevalent. Conclusions: Performing RET screening in Ceará allowed us to identify a different mutation profile in this region compared with other areas. RET screening also enabled the diagnosis of a significant number of hereditary MTC patients who were initially classified as sporadic disease patients and benefited their relatives, who were unaware of the risks and the consequences of bearing a RET mutation.

SUMMARY Identification of the correct etiology of diabetes brings important implications for clinical management. In this report, we describe a case of a 4-year old asymptomatic girl with diabetes since age 2, along with several individuals in her family with different etiologies for hyperglycemia identified in youth. Genetic analyses were made by Sanger sequencing, laboratory measurements included HbA1c, lipid profile, fasting C-peptide, pancreatic auto-antibodies (glutamic acid decarboxylase [GAD], Islet Antigen 2 [IA-2], and anti-insulin). We found a Gly178Ala substitution in exon 5 of GCK gene in three individuals co-segregating with diabetes, and type 1 diabetes was identified in two other individuals based on clinical and laboratory data. One individual with previous gestational diabetes and other with prediabetes were also described. We discuss difficulties in defining etiology of hyperglycemia in youth in clinical practice, especially monogenic forms of diabetes, in spite of the availability of several genetic, laboratory, and clinical tools.

ABSTRACT Thyroglobulin (Tg) is the major glycoprotein produced by the thyroid gland, where it serves as a template for thyroid hormone synthesis and as an intraglandular store of iodine. Measurement of Tg levels in serum is of great practical importance in the follow-up of differentiated thyroid carcinoma (DTC), a setting in which elevated levels after total thyroidectomy are indicative of residual or recurrent disease. The most recent methods for serum Tg measurement are monoclonal antibody-based and are highly sensitive. However, major challenges remain regarding the interpretation of the results obtained with these immunometric methods, particularly in patients with endogenous antithyroglobulin antibodies or in the presence of heterophile antibodies, which may produce falsely low or high Tg values, respectively. The increased prevalence of antithyroglobulin antibodies in patients with DTC, as compared with the general population, raises the very pertinent possibility that tumor Tg may be more immunogenic. This inference makes sense, as the tumor microenvironment (tumor cells plus normal host cells) is characterized by several changes that could induce posttranslational modification of many proteins, including Tg. Attempts to understand the structure of Tg have been made for several decades, but findings have generally been incomplete due to technical hindrances to analysis of such a large protein (660 kDa). This review article will explore the complex structure of Tg and the potential role of its marked heterogeneity in our understanding of normal thyroid biology and neoplastic processes.

Educational change initiatives, whether they involve the implementation of new policies or curriculum reforms, often fail to reach the level of teachers' classroom practices. In the search for explanations, numerous studies have either characterized teachers as resistant to change or focused on how schools' workplace conditions have failed to influence teacher change. This study draws attention to these issues by investigating teachers' perceptions of the degree to which a national curriculum reform has brought about changes in their teaching. The study also proposes a model of how teachers' perceptions of school-based conditions (i.e., school leadership, teacher collaboration, and development of school-based curricula) are related to teachers' general orientations towards change and teachers' perceptions of change as a result of the reform. The participants were 738 Norwegian teachers in public primary and lower secondary schools, and the data were analyzed using descriptive data, correlation analysis and structural equation modeling (SEM).The results indicate that the overall influence of the reform on teachers' teaching was moderate. The results also revealed positive relationships between teachers' perceptions of school-based conditions, teachers' general orientations towards change and the degree of perceived reform changes in teachers' teaching. However, a surprising finding is that school leadership and teacher collaboration were only indirectly related to teachers' perceptions of reform changes, mediated through teachers' general orientations towards change. The article highlights important relationships between educational reform, school-based conditions and teachers. It also contributes knowledge that is relevant to consider for successful reform of schools and education.

Genetic testing for germline RET pathogenic variants, which cause the Multiple Endocrine Neoplasia Type 2 (MEN2) syndrome, has become crucial in managing patients with medullary thyroid carcinoma (MTC). Classically, RET heterozygous missense pathogenic variants are transmitted in a Mendelian autosomal dominant pattern, of which germline/gonadal mosaicism has never been reported. We report the novel occurrence of a MEN2A patient's family in which the siblings inherited three different RET 634 genotypes: wild type (p.Cys634), p.Cys634Gly or p.Cys634Arg heterozygous pathogenic variants. We hypothesized that germline/gonadal mosaicism, derived from an inherited + early somatic mutation in the mother or a double de novo mutation during maternal embryogenesis, led to this rare event in the RET gene. Exome analysis of the proband's deceased mother's paraffin‐embedded thyroid tissue confirmed the three nucleotides in the same 634 codon position. For the first time, we describe germline/gonadal mosaicism in RET, generating a second pathogenic amino acid change in the same codon causing MEN2A. Our finding shows that RET parental mosaicism, confirmed by somatic exome sequencing, might explain discrepant genotype cases in siblings with inherited cancers. [ABSTRACT FROM AUTHOR]

The reaction of organoberyllium compounds with hexaphenylcarbodiphosphorane yields mono‐ortho‐beryllated complexes, which feature a double dative Be=C bond. The bonding situation in these compounds together with a simple carbodiphosphorane and an N‐heterocyclic carbene adduct was analysed with energy decomposition analysis in combination with natural orbital for chemical valence as well as with quantum theory of atoms‐in‐molecules. Furthermore, the driving forces accountable for mono‐ortho‐beryllation were elucidated along with the reactivity of the Be=C bond. [ABSTRACT FROM AUTHOR]

Introduction: Preventing potential foreign animal diseases is a high priority, with re-emerging threats such as African Swine Fever emerging close to North American borders. The Secure Pork Supply (SPS) plan provides a voluntary framework for swine producer biosecurity planning and disease outbreak preparedness. However, biosecurity knowledge varies greatly among swine veterinarians, managers, and caretakers within the industry, which impacts the understanding, quality, implementation and biosecurity plan agreements with the SPS guidelines unless review procedures and quality control mechanisms are in place. Therefore, this study aimed to describe and identify the level of biosecurity planning agreements between producer-and/or swine veterinarian- made biosecurity plans for commercial swine sites and the SPS plan guidelines during a review process. Material and methods: Biosecurity maps (N = 368) and written plans (N = 247) were obtained from six Midwest swine companies/veterinary clinics. Maps were evaluated on accuracy and placement of mandatory map features based on SPS guidelines, and discrepancies between the development of producer- made biosecuritymaps and written biosecurity plans.Multivariablemixed logistic regression analyses were conducted to identify swine farms. Future of producers made plants. [ABSTRACT FROM AUTHOR]