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Abstract Genome-wide association studies (GWAS) have become well-powered to detect loci associated with telomere length. However, no prior work has validated genes nominated by GWAS to examine their role in telomere length regulation. We conducted a multi-ancestry meta-analysis of 211,369 individuals and identified five novel association signals. Enrichment analyses of chromatin state and cell-type heritability suggested that blood/immune cells are the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6 or POP5 and demonstrated that both lengthened telomeres. CRISPR/Cas9 deletion of the predicted causal regions in K562 blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5. Our results demonstrate the utility of telomere length GWAS in the identification of telomere length regulation mechanisms and validate KBTBD6 and POP5 as genes affecting telomere length regulation.

BackgroundClassical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care.MethodsA loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated.ResultsThree primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles.ConclusionThe results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Abstract Background Oral health plays a role in overall health, indicating the need to identify barriers to accessing oral care. The objective of this study was to identify barriers to accessing oral health care and examine the association between socioeconomic, psychosocial, and physical measures with access to oral health care among older Canadians. Methods A cross-sectional study was conducted using data from the Canadian Longitudinal Study on Aging (CLSA) follow-up 1 survey to analyze dental insurance and last oral health care visit. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between socioeconomic, psychosocial, and physical measures with access to oral care, measured by dental insurance and last oral health visit. Results Among the 44,011 adults included in the study, 40% reported not having dental insurance while 15% had not visited an oral health professional in the previous 12 months. Several factors were identified as barriers to accessing oral health care including, no dental insurance, low household income, rural residence, and having no natural teeth. People with an annual income of

Nitrofurantoin, a broad-spectrum nitrofuran class antibiotic, is applied as a first-line antibiotic in treating human urinary tract infections (UTIs) due to its great efficacy and high achievable concentration. The interest in using this antibiotic in companion animals has increased due to the growing demand for effective antibiotics to treat UTIs caused by multidrug-resistant bacteria. Currently, the susceptibility interpretations for nitrofurantoin are based on the breakpoints set for humans, while the canine-specific breakpoints are still unavailable. In this study, we assessed the concentration of nitrofurantoin reaching the dog’s urine using the recommended oral dosing regimen. In addition, we examined the efficacy of this breakpoint concentration against the common canine UTI pathogens, Escherichia coli, Staphylococcus pseudintermedius, and Enterococcus faecium. Eight experimental beagle dogs were treated with ~5 mg/kg of nitrofurantoin macrocrystal PO 8qh for 7 days. The urine samples were collected via cystocentesis at 2, 4, and 6 h after administration on day 2 and day 7 and used to quantify nitrofurantoin concentrations by ultra-high performance liquid chromatography. The results showed that 26.13–315.87 μg/mL nitrofurantoin was detected in the dogs’ urine with a mean and median concentration of 104.82 and 92.75 μg/mL, respectively. Additionally, individual dogs presented with urinary nitrofurantoin concentrations greater than 64 μg/mL for at least 50% of the dosing intervals. This concentration efficiently killed E. coli, and S. pseudintermedius, but not E. faecium strains carrying an MIC90 value equal to 16, 16, and 128 μg/mL, respectively. Taken together, these results suggest that the value of 64 μg/mL may be set as a breakpoint against UTI pathogens, and nitrofurantoin could be an effective therapeutic drug against E. coli and S. pseudintermedius for canine UTIs.

African swine fever virus (ASFV) currently represents the biggest threat to the porcine industry worldwide, with high economic impact and severe animal health and welfare concerns. Outbreaks have occurred in Europe and Asia since ASFV was reintroduced into the continent in 2007 and, in 2021, ASFV was detected in the Caribbean, raising alarm about the reemergence of the virus in the Americas. Given the lack of vaccines against ASFV, control of the virus relies on molecular surveillance, which can be delayed due to the need for sample shipment to specialized laboratories. Isothermal PCR techniques, such as LAMP, have become increasingly attractive as point-of-care diagnostic tools given the minimal material expense, equipment, and training required. The present study aimed to develop a LAMP assay for the detection of ASFV. Four LAMP primer sets were designed, based on a consensus sequence for the ASFV p72 gene, and were tested using a synthetic plasmid containing the cloned ASFV p72 target gene as a positive control. Two primer sets, were selected for further validation, given their very short time for amplification. Both primer sets showed thermal stability, amplifying the ASFV DNA at temperatures between 60-70°C and proved to have an analytical limit of detection as low as one ASFV-plasmid DNA copy/µL, using both fluorometric and colorimetric methods. The selected primers did not yield false positive or cross reactive results with other common swine pathogens, showing high specificity. Testing of DNA-spiked samples showed that LAMP amplification was not affected by the nature of the matrices, including oral fluids, tonsils, blood, or rectal swabs. The primer sets were able to detect the two more prevalent ASFV genotypes in the field. Taken together, the results show that ASFV-LAMP-BG2 and ASFV-LAMP-BG3 would be a useful tool for rapid, highly sensitive on-site diagnostic testing.

Three adult littermates were diagnosed with Brucella canis, two of which were diagnosed with discospondylitis. The first littermate, a 2-year-old spayed-female Labrador Retriever, was evaluated for progressive episodes of cervical pain, lethargy, reported circling to the right, and a right-sided head tilt. Magnetic resonance imaging (MRI) of the cervical spine revealed changes consistent with discospondylitis at C6-C7. MRI of the brain was unremarkable and cerebrospinal fluid analysis was declined. Brucella spp. was isolated from aerobic and Brucella blood cultures. PCR performed on the isolate identified Brucella canis and indirect fluorescent antibody (IFA) testing for Brucella canis also confirmed the species. Patient #1 was treated with doxycycline and marbofloxacin for 1 year. Clinical signs returned 2-years after diagnosis. Following the diagnosis of patient #1, a known littermate (patient #2) was tested for Brucella canis. Patient #2 was 2 years old and asymptomatic at the time of diagnosis. Aerobic and Brucella spp. cultures, PCR, and IFA were obtained and were diagnostic for Brucella canis. A 6-month course of marbofloxacin and doxycycline was implemented. The patient remained PCR positive following 4 months of treatment and repeat cultures were planned following 6 months of treatment; however, the patient was lost to follow-up. A third littermate (patient #3) was identified by the family of patient #1. Patient #3 was evaluated at 18 months of age for a 6-month history of progressive lumbosacral pain. Spinal radiographs revealed discospondylitis of the C3-C4, T12-T13, and L7-S1 vertebral endplates. Computed tomography (CT) of the lumbosacral spine was also consistent with discospondylitis at L7-S1. Brucella canis serologic testing consisting of rapid slide agglutination test, 2ME-rapid slide agglutination test, and cytoplasmic agar gel immunodiffusion was positive. Enrofloxacin was administered for 7 months and was discontinued thereafter based on radiographic evidence of healing and resolution of clinical signs. Although Brucella canis is not a rare disease in dogs, the documentation of two out of three adult littermates with associated discospondylitis is an interesting feature. In addition, this report highlights available diagnostic and treatment options, as each patient was managed differently based on clinical signs and the preference of the managing clinician.

Abstract Background More than half of adolescents have jobs in summer or sometime during the year. While employers are ultimately responsible for their safety, parents are often important in helping their children navigate the work environment. Our study examines the attitudes, beliefs and types of involvement parents have in their children’s work. Methods We modeled a telephone survey of 507 English-speaking parents of working adolescents in Ontario, Canada on a US study and examined their perspectives, comparing to earlier findings from the U.S. parents. Results Most Ontario parents helped their teens consider questions to ask about work, for example, work hours (90.7%) and job tasks (78.2%) and fewer about workplace safety (57.9%). Parents overall were concerned about their teens, especially younger teens, getting behind on schoolwork (69.3%), being rushed on the job (60.1%) and doing hazardous tasks (58.3%) or working alone (51.9%), or being at work during a robbery (74.5%). Parents of 14–17-year-old daughters were more concerned about their child being assaulted than were parents of sons (62.4% vs. 51.4%), particularly if the teen was in the 18–19 age group (74.3% vs. 52.5%). Half the parents indicated 10–19 h per week was the right amount of work time for their teen, and most agreed that laws should limit the number of hours of youth work. Conclusions Overall, Ontario parents appear to be more concerned about the safety and also more involved in the work of their adolescent children than U.S. parents previously surveyed. Parents are engaged with their children about their work and may serve as valuable assets to helping to advocate for safe work policies and environments.

The emergence of antimicrobial resistance (AMR) in dogs constitutes a threat to animal and human health. There is a lack of studies in Illinois that evaluated the prevalence of AMR among urinary bacterial pathogens. In the study, we included 803 isolates (299 Gram-positive and 504 Gram-negative) that were isolated from 2,583 canine urine samples submitted to the Veterinary Diagnostic Laboratory, the University of Illinois between 2019 and 2020 from dogs suspected of urinary tract infections (UTI). The most common Gram-positive isolates included Staphylococcus pseudintermedius (17.93%), Enterococcus faecalis (9.46%), Streptococcus canis (6.10%), and Enterococcus faecium (3.74%), while Gram-negative isolates included Escherichia coli (45.58%), Proteus mirabilis (11.08%), Klebsiella pneumoniae (3.11%), and Pseudomonas aeruginosa (2.99%). Among the Gram-positive isolates, Staphylococcus pseudintermedius isolates showed a very high prevalence of resistance to penicillin (56.94%), a high prevalence of resistance to trimethoprim-sulfamethoxazole (31.94%), enrofloxacin (29.17%), and oxacillin (27.08%). Among Gram-negative bacteria, Escherichia coli isolates showed a high prevalence of resistance to ampicillin (31.42%). Considering the high prevalence of resistance to antimicrobials commonly used to treat UTI in dogs, urine samples should be collected for bacterial culture and susceptibility testing before treatment initiation to prevent treatment failures and the development of multidrug resistance. Given the possibility of zoonotic transmission of antimicrobial-resistant bacteria, veterinarians when treating UTI cases, should inform dog owners of the potential transmission risk.

BackgroundHigh-prevalence childhood mental health problems like early-onset disruptive behavior problems (DBPs) pose a significant public health challenge and necessitate interventions with adequate population reach. The treatment approach of choice for childhood DBPs, namely evidence-based parenting intervention, has not been sufficiently disseminated when relying solely on staff-delivered services. Online-delivered parenting intervention is a promising strategy, but the cost minimization of this delivery model for reducing child DBPs is unknown compared with the more traditional staff-delivered modality. ObjectiveThis study aimed to examine the cost-minimization of an online parenting intervention for childhood disruptive behavior problems compared with the staff-delivered version of the same content. This objective, pursued in the context of a randomized trial, made use of cost data collected from parents and service providers. MethodsA cost-minimization analysis (CMA) was conducted comparing the online and staff-delivered parenting interventions. Families (N=334) with children 3-7 years old, who exhibited clinically elevated disruptive behavior problems, were randomly assigned to the two parenting interventions. Participants, delivery staff, and administrators provided data for the CMA concerning family participation time and expenses, program delivery time (direct and nondirect), and nonpersonnel resources (eg, space, materials, and access fee). The CMA was conducted using both intent-to-treat and per-protocol analytic approaches. ResultsFor the intent-to-treat analyses, the online parenting intervention reflected significantly lower program costs (t168=23.2; P

Summary: Chronic graft-versus-host disease (cGVHD) targets include the oral mucosa and salivary glands after allogeneic hematopoietic stem cell transplant (HSCT). Without incisional biopsy, no diagnostic test exists to confirm oral cGVHD. Consequently, therapy is often withheld until severe manifestations develop. This proteomic study examined saliva and human salivary gland for a biomarker profile at first onset of oral cGVHD prior to initiation of topical steroid therapy. Whole saliva collected at onset of biopsy-proven oral GVHD was assessed using liquid chromatography–coupled tandem mass spectrometry with identification of 569 proteins, of which 77 significantly changed in abundance. ZG16B, a secretory lectin protein, was reduced 2-fold in oral cGVHD saliva (p

Dermatophytosis is a common and highly contagious zoonotic skin disease in companion animals. This disease is a major concern in geographical areas that contain large numbers of stray animal populations. Numerous surveys on dermatophytosis among stray animal populations worldwide range between 27% to 50%. In recent years, the US territory of Puerto Rico was impacted by several natural disasters such as hurricanes, which has led to a large increase of abandonment cases and an increase in the stray animal population. Due to this, large low-cost spay/neuter clinics and trap-neuter-release programs have become a more common practice on the island. During these events, veterinary staff are exposed to multiple animals with no health history, and therefore, zoonotic diseases are of concern. The aim of this study was to provide information regarding the presence of dermatophyte species in symptomatic and asymptomatic stray dogs and cats in a region of Puerto Rico. Hair samples were collected from 99 stray animals with and without dermatological clinical signs. The hair samples were cultured on plates containing rapid sporulation medium and dermatophyte test medium. All cultures were evaluated microscopically to confirm the presence of dermatophytes. Then, all dermatophytes were further evaluated with MALDI-TOF MS to compare both diagnostic tests. A total of 19 animals (19%) were positive for dermatophyte growth. Of these animals, 18/19 were infected with M. canis and 1/19 with Trichophyton spp. Animals with clinical lesions were positive only 13.5% of the time compared to asymptomatic animals, who were positive in 36% of the sample population. All 19 dermatophytes (100%) diagnosed with microscopic evaluation were confirmed with MALDI-TOF MS. Our results indicate that there is a prevalence of 19% of dermatophytosis among the stray dog and cat population of the southeastern coast of the island.

ABSTRACT Staphylococcus pseudintermedius is a major canine pathogen but also occasionally colonizes and infects humans. Multidrug-resistant methicillin-resistant S. pseudintermedius (MDR MRSP) strains have emerged globally, making treatment and control of this pathogen challenging. Sequence type 71 (ST71), ST68, and ST45 are the most widespread and successful MDR MRSP clones. The potential genetic factors underlying the clonal success of these and other predominant clones remain unknown. Characterization of the pangenome, lineage-associated accessory genes, and genes acquired through horizontal gene transfer from other bacteria is important for identifying such factors. Here, we analyzed genome sequence data from 622 S. pseudintermedius isolates to investigate the evolution of pathogenicity across lineages. We show that the predominant clones carry one or more lineage-associated virulence genes. The gene encoding staphylococcal protein A (SpA), a key virulence factor involved in immune evasion and a potential vaccine antigen, is deleted in 62% of isolates. Most importantly, we have discovered that the spa locus is a hot spot for recombination and horizontal gene transfer in S. pseudintermedius, where genes related to restriction modification, prophage immunity, mercury resistance, and nucleotide and carbohydrate metabolism have been acquired in different lineages. Our study also establishes that ST45 is composed of two distinct sublineages that differ in their accessory gene content and virulence potential. Collectively, this study reports several previously undetected lineage-associated genetic factors that may have a role in the clonal success of the major MDR MRSP clones. These data provide a framework for future experimental studies on S. pseudintermedius pathogenesis and for developing novel therapeutics against this pathogen. IMPORTANCE Staphylococcus pseudintermedius is a major canine pathogen but can also occasionally infect humans. Identification of genetic factors contributing to the virulence and clonal success of multidrug-resistant S. pseudintermedius clones is critical for the development of therapeutics against this pathogen. Here, we characterized the genome sequences of a global collection of 622 S. pseudintermedius isolates. We show that all major clones, besides carrying core virulence genes, which are present in all strains, carry one or more lineage-specific genes. Many of these genes have been acquired from other bacterial species through a horizontal gene transfer mechanism. Importantly, we have discovered that the staphylococcal protein A gene (spa), a widely used marker for molecular typing of S. pseudintermedius strains and a potential vaccine candidate antigen, is deleted in 62% of strains. Furthermore, the spa locus in S. pseudintermedius acts as a reservoir to accumulate lineage-associated genes with adaptive functions.

To examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ∆N-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that de-regulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-∆1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

Leptospirosis is recognized as the most globally widespread reemerging zoonosis and represents a serious threat for both human and animal health. Indeed, leptospirosis is linked to more than 60,000 human deaths per year and to incalculable economic burden as consequence of medical treatment costs and livestock loss. The increasing number of reports from species of pathogenic Leptospira spp. group II causing disease in both humans and animals constitutes an additional concern to the complex epidemiology of this zoonotic agent. Diagnostic methods based on qPCR have improved the diagnosis of Leptospira spp. in terms of cost, time, and reliability, but most of the validated assays fail to detect species from the pathogenic group II. Hence, the current study was aimed to develop and validate a novel multiplex qPCR to enable the specific and selective detection of the whole group of infectious Leptospira spp., including both pathogenic groups I and II and moreover, selectively discriminate between them. To fit the “fitness of purpose” for the specific detection of infectious Leptospira spp. and further discrimination between both pathogenic groups three target regions on the 16S RNA gene were selected. These targets facilitated a broad and selective spectrum for the detection of all infectious Leptospira spp. with the exclusion of all saprophytic groups and the novel clade of environmental Leptospira spp. The analytical sensitivity (ASe) showed by the new assay also enables a wide window of detection for the agent at different stages of infection since the assay was able to efficiently detect at 95% of confidence ∼5 leptospires/reaction. From the evaluation of the analytical specificity (ASp) by in silico and in vitro approaches, it was congruently revealed that the primers and probes selected only recognized the specific targets for which the assay was intended. Bayesian latent class analysis of performance of the new assay on 684 clinical samples showed values of diagnostic sensitivity of 99.8% and diagnostic specificity of 100%. Thus, from the evaluation of the analytical and diagnostic parameters, the new multiplex qPCR assay is a reliable method for the diagnosis of Leptospira spp.

ABSTRACT Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of soft tissue infections in dogs and occasionally infects humans. Hypervirulent multidrug-resistant (MDR) MRSP clones have emerged globally. The sequence types ST71 and ST68, the major epidemic clones of Europe and North America, respectively, have spread to other regions. The genetic factors underlying the success of these clones have not been investigated thoroughly. Here, we performed a comprehensive genomic analysis of 371 S. pseudintermedius isolates to dissect the differences between major clonal lineages. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, restriction-modification (RM), and CRISPR/Cas systems differs significantly among MRSP clones. The isolates with GyrA+GrlA mutations, conferring fluoroquinolone resistance, carry more of these genes than those without GyrA+GrlA mutations. ST71 and ST68 clones carry lineage-specific prophages with genes that are likely associated with their increased fitness and virulence. We have discovered that a prophage, SpST71A, is inserted within the comGA gene of the late competence operon comG in the ST71 lineage. A functional comG is essential for natural genetic competence, which is one of the major modes of horizontal gene transfer (HGT) in bacteria. The RM and CRISPR/Cas systems, both major genetic barriers to HGT, are also lineage specific. Clones harboring CRISPR/Cas or a prophage-disrupted comG exhibited less genetic diversity and lower rates of recombination than clones lacking these systems. After Listeria monocytogenes, this is the second example of prophage-mediated competence disruption reported in any bacteria. These findings are important for understanding the evolution and clonal expansion of MDR MRSP clones. IMPORTANCE Staphylococcus pseudintermedius is a bacterium responsible for clinically important infections in dogs and can infect humans. In this study, we performed genomic analysis of 371 S. pseudintermedius isolates to understand the evolution of antibiotic resistance and virulence in this organism. The analysis covered significant reported clones, including ST71 and ST68, the major epidemic clones of Europe and North America, respectively. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, and horizontal gene transfer differs among clones. ST71 and ST68 carry prophages with novel virulence and antibiotic resistance genes. Importantly, site-specific integration of a prophage, SpST71A, has led to the disruption of the genetic competence operon comG in ST71 clone. A functional comG is essential for the natural uptake of foreign DNA and thus plays an important role in the evolution of bacteria. This study provides insight into the emergence and evolution of antibiotic resistance and virulence in S. pseudintermedius, which may help in efforts to combat this pathogen.

Abstract Background Continuous-flow left ventricular assist devices (CF-LVAD) have become the standard of care for patients with end stage heart failure. Device reliability has increased, bringing the potential for VAD, compared to transplant, into debate. However, complications continue to limit VADs as first line therapy. Bleeding is a major morbidity. A debate exists as to the difference in bleeding profile between the major centrifugal and axial flow devices. We hypothesized that there would be similar adverse bleeding event profiles between the 2 major CF-LVADs. Methods We retrospectively investigated isolated CF LVADs performed at our institution between July 2010 and July 2015: HeartMateII (HMII, n = 105) and HeartWare (HVAD, n = 34). We reviewed demographic, perioperative and short- and long-term outcomes. Results There was no significant difference in demographics or comorbidities. There was a low incidence of gastrointestinal (GI) bleed 3.9% in HMII and 2.9% in HVAD (p = 0.78). Preoperatively, the cohorts did not differ in coagulation measures (p = 0.95). Within the post-operative period, there was no difference in product transfusion: red blood cells (p = 0.10), fresh frozen plasma (p = 0.19), and platelets (p = 0.89). Post-operatively, a higher but not significantly different number of HMII patients returned to the operating room for bleeding (n = 27) compared to HVAD (n = 6, p = 0.35). There was no difference in rates of stroke (p = 0.65), re-intubation (p = 0.60), driveline infection (p = 0.05), and GI bleeding (p = 0.31). The patients had equivalent ICU LOS (p = 0.86) and index hospitalization LOS (p = 0.59). Conclusion We found no difference in the rate of bleeding complications between the current commercially available axial and centrifugal flow devices.

The effect of biochar addition on the anaerobic digestion (AD) of food waste was evaluated. From the five biochars tested, Fe, Co, Ni, and Mn were leached in very small quantities (

Introduction: A youth’s emergency department (ED) visit for suicidal behaviors or ideation provides an opportunity to counsel families about securing medications and firearms (i.e., lethal means counseling). Methods: In this quality improvement project drawing on the Counseling on Access to Lethal Means (CALM) model, we trained 16 psychiatric emergency clinicians to provide lethal means counseling with parents of patients under age 18 receiving care for suicidality and discharged home from a large children’s hospital. Through chart reviews and follow-up interviews of parents who received the counseling, we examined what parents recalled, their reactions to the counseling session, and actions taken after discharge. Results: Between March and July 2014, staff counseled 209 of the 236 (89%) parents of eligible patients. We conducted follow-up interviews with 114 parents, or 55% of those receiving the intervention; 48% of those eligible. Parents had favorable impressions of the counseling and good recall of the main messages. Among the parents contacted at follow up, 76% reported all medications in the home were locked as compared to fewer than 10% at the time of the visit. All who had indicated there were guns in the home at the time of the visit reported at follow up that all were currently locked, compared to 67% reporting this at the time of the visit. Conclusion: Though a small project in just one hospital, our findings demonstrate the feasibility of adding a counseling protocol to the discharge process within a pediatric psychiatric emergency service. Our positive findings suggest that further study, including a randomized control trial in more facilities, is warranted.

The quantity of the intra-erythrocytic deoxyhemoglobin S (Hb S) affects the level of protection against malaria and also the sickling phenomenon. This study reports on significantly lower concentration of Hb S in females than males. Data came from 350 children, aged 12-47 months who participated in a phase 2b malaria vaccine trial. Hemoglobinopathy and G6PD deficiency typing was necessary to ascertain equal representation of these malaria protective traits across the vaccine cohorts. Hemoglobin types (HbAA, HbAS) and % Hb S were evaluated by HPLC. Alpha thalassemia (alpha-thal) and G6PD genotypes were evaluated by PCR. The overall prevalence for HbAS was 20%, 46% for 3 alpha genes and 10% for 2 alpha genes and 14% for G6PD A-. More females of HbAS/αα/αα genotype had low Hb S than males and had mean % Hb S of 37.5% ± 5.4 SD, compared to 42.0% ± 2.5 SD in males of same genotype (P = 0.018). Consistent with reduction of the malaria protective Hb S in females, parasite load in females was nearly twice that of males but the difference was not statistically significant. The X-chromosome linked G6PD deficiency did not influence the level of Hb S. We conclude that, the low Hb S in these females explains the resultant higher malaria parasite load. We speculate that the low Hb S in females could also explain observations suggesting that the sickling phenomenon tends to be less severe in females than males.

Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago.

RTS,S, a candidate vaccine for malaria, is a recombinant protein expressed in yeast containing part of the circumsporozoite protein (CSP) sequence of 3D7 strain of Plasmodium falciparum linked to the hepatitis B surface antigen in a hybrid protein. The RTS,S antigen is formulated with GSK Biologicals' proprietary Adjuvant Systems AS02(A) or AS01(B). A recent trial of the RTS,S/AS02(A) and RTS,S/AS01(B) vaccines evaluated safety, immunogenicity and impact on the development of parasitemia of the two formulations. Parasite isolates from this study were used to determine the molecular impact of RTS,S/AS02(A) and RTS,S/AS01(B) on the multiplicity of infection (MOI) and the csp allelic characteristics of subsequent parasitemias.The distribution of csp sequences and the MOI of the infecting strains were examined at baseline and in break-through infections from vaccinated individuals and from those receiving a non-malarial vaccine.The study was conducted in Kombewa District, western Kenya.Semi-immune adults from the three study arms provided isolates at baseline and during break-through infections.Parasite isolates used for determining MOI and divergence of csp T cell-epitopes were 191 at baseline and 87 from break-through infections.Grouping recipients of RTS,S/AS01(A) and RTS,S/AS02(B) together, vaccine recipients identified as parasite-positive by microscopy contained significantly fewer parasite genotypes than recipients of the rabies vaccine comparator (median in pooled RTS,S groups: 3 versus 4 in controls, P = 0.0313). When analyzed separately, parasitaemic individuals in the RTS,S/AS01(B) group, but not the RTS,S/AS02(A) group, were found to have significantly fewer genotypes than the comparator group. Two individual amino acids found in the vaccine construct (Q339 in Th2R and D371 in Th3R) were observed to differ in incidence between vaccine and comparator groups but in different directions; parasites harboring Q339 were less common among pooled RTS,S/AS vaccine recipients than among recipients of rabies vaccine, whereas parasites with D371 were more common among the RTS,S/AS groups.It is concluded that both RTS,S/AS vaccines reduce multiplicity of infection. Our results do not support the hypothesis that RTS,S/AS vaccines elicit preferential effects against pfcsp alleles with sequence similarity to the 3D7 pfcsp sequence employed in the vaccine construct.

This paper documents the outcomes of the first 174 patients enrolled in a recently established community-based antiretroviral (ARV) treatment programme. Many people are living with HIV/AIDS and are in need of ARVs. Access to ARVs can pose many logistical difficulties for poor and sick persons, particularly in rural areas. This communitybased treatment programme aimed to overcome these challenges, providing transport and nursing care, as well as actively seeking persons too sick to leave their homes. This led to the inclusion of many persons with advanced HIV in the treatment population. The study used routinely collected CD4 counts, viral loads and patient observation. The results show that the majority of patients responded well to treatment. There was higher mortality among the population with advanced disease, but a considerable proportion responded well. Only 4 patients were lost to follow-up. This study details how community-based ARV programmes can be useful adjuncts to the facility-based system. Effective treatment programmes must address the needs of the communities they serve.

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase-some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR-/- and Emumyc+mTR-/-) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR-/- tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR-/- cells that had short telomeres. Using mouse mTR+/- and human hTERT+/- primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length.

We developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae Nanopore allows long-read sequencing through the telomere, through the subtelomere, and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end-specific telomere lengths that were stable over 120 cell divisions. These stable chromosome-specific telomere lengths may be explained by slow clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosome end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Y' sequences, tel1Δ rif1Δ double mutants had a very small, but significant, increase in telomere length compared with the tel1Δ single mutant, suggesting an influence of Y' elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2Δ) and found the minimal telomere length to be ∼75 bp. In these est2Δ mutants, there were apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2Δ rad52Δ double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at ∼5 bp per generation. This new method gives quantitative, high-resolution telomere length measurement at each individual chromosome end and suggests possible new biological mechanisms regulating telomere length.

Telomere length regulation is essential for cell viability in eukaryotes. While many pathways that affect telomere length are known, we do not yet have a complete understanding of the mechanism of length regulation. To identify new pathways that might regulate telomere length, we carried out a genetic screen in yeast and identified the cyclin-dependent kinase complex Bur1/2 as a regulator of telomere length. Mutations in either BUR1 cyclin-dependent kinase or the associated BUR2 cyclin resulted in short telomeres. This regulation did not function through the known role of BUR1 in regulating histone modification as bur1∆ set2∆ and bur2∆ set2∆ double mutants rescued cell growth but did not rescue the telomere shortening effects. We found that both bur1∆ and bur2∆ set2∆ were also defective in de novo telomere addition, and deletion of SET2 did also not rescue this elongation defect. The Bur1/2 cyclin-dependent kinase regulates transcription of many genes. We found that TLC1 RNA levels were reduced in bur2∆ set2∆ mutants; however, overexpression of TLC1 restored the transcript levels but did not restore de novo telomere elongation or telomere length. These data suggest that the Bur1/2 kinase plays a role in telomere elongation separate from its role in transcription of telomerase components. Dissecting the role of the Bur1/2 kinase pathway at telomeres will help complete our understanding of the complex network of telomere length regulation.

In budding yeast, Rif1 negatively regulates telomere length, but the mechanism of this regulation has remained elusive. Previous work identified several functional domains of Rif1, but none of these has been shown to mediate telomere length. To define Rif1 domains responsible for telomere regulation, we localized truncations of Rif1 to a single specific telomere and measured telomere length of that telomere compared to bulk telomeres. We found that a domain in the N-terminus containing HEAT repeats, Rif1177-996, was sufficient for length regulation when tethered to the telomere. Charged residues in this region were previously proposed to mediate DNA binding. We found that mutation of these residues disrupted telomere length regulation even when Rif1 was tethered to the telomere. Mutation of other conserved residues in this region, which were not predicted to interact with DNA, also disrupted telomere length maintenance, while mutation of conserved residues distal to this region did not. Our data suggest that conserved amino acids in the region from 436 to 577 play a functional role in telomere length regulation, which is separate from their proposed DNA binding function. We propose that the Rif1 HEAT repeats region represents a protein-protein binding interface that mediates telomere length regulation.

Background: Parenting strategies such as communicating clear expectations, providing calm directions, and teaching specific skills can strengthen young children's social-emotional development. Parenting programs for children with disruptive behavior often emphasize gaining compliance via effective directives, and less on how to facilitate child skill acquisition or on effective parenting for differing situations and task demands. Objective: We aimed to study how parenting strategies and associated child behavior vary by situational contexts. Specifically, we focused on the differential use of directives and guidance during different tasks. Method: This observation study utilized a microsocial coding system, the Parent-Child Play Task Observation System (PCPTOS), to closely examine parent and child interactions in multiple analogue task situations. The study drew on pre-intervention data for 224 parent-child dyads who participated in a parenting-focused intervention trial for children ages 3-7 who presented elevated levels of disruptive behaviors. Results: Interrater reliabilities were very good to excellent. Parents used directives more frequently during the clean-up task and guidance more frequently during the teaching task compared to the other tasks. Associations of parent use of directives and guidance with child behavior and affect differed by task. Observed parent directives were associated with child disruptive behavior during each task, whereas parent guidance was negatively associated with child disruptive behavior and positively associated with child positive affect during the teaching task. Conclusions: Parenting strategies that are well matched to the situational context and proactively consider task demands are more likely to facilitate children's social-emotional development.

To examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ∆N-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that de-regulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-∆1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

Previous models suggested that regulation of telomere length in Saccharomyces cerevisiae by Tel1(ATM) and Mec1(ATR) would parallel the established pathways regulating the DNA damage response. Here, we provide evidence that telomere length regulation differs from the DNA damage response in both the Tel1 and Mec1 pathways. We found that Rad53 mediates a Mec1 telomere length regulation pathway but is dispensable for Tel1 telomere length regulation, whereas in the DNA damage response, Rad53 is regulated by both Mec1 and Tel1 Using epistasis analysis with a Tel1 hypermorphic allele, Tel1-hy909, we found that the MRX complex is not required downstream of Tel1 for telomere elongation but is required downstream of Tel1 for the DNA damage response. Our data suggest that nucleolytic telomere end processing is not a required step for telomerase to elongate telomeres.

TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex.

Aims: Judicious antimicrobial use in companion animal practice is critical for maintaining the effectiveness of antimicrobial agents against bacterial infections and reducing the selection of antimicrobial‐resistant bacteria. This study aimed to provide insights into companion animal veterinarians' antimicrobial treatment recommendations for common bacterial infections in dogs and cats and describe the factors influencing their prescription choices. Methods and Results: An online survey using QualtricsXM® software was administered between September and November 2022 to companion animal veterinarians who were Illinois State Veterinary Medical Association members. Descriptive and text analyses were conducted to assess the participants' responses. A total of 78 surveys were included in the analysis. Skin infections were ranked as the most common bacterial infections for which veterinarians prescribed antimicrobial agents, followed by ear, urinary tract, respiratory, and enteric infections. The severity of clinical symptoms and the results of bacterial culture and susceptibility tests were the most influential factors for veterinarians when making antimicrobial prescription choices. Veterinarians were aware of the current antimicrobial prescription guideline recommendations when prescribing antimicrobials empirically to nine hypothetical scenarios of bacterial infections. According to the results of the text analysis that assessed veterinarians' responses to an open‐ended question, regarding their challenges when prescribing antimicrobial agents, the pairwise correlation of word frequencies within each response showed the highest correlations between the words 'owner' and 'compliance', 'administration' and 'route', 'cost' and 'culture', and 'patients' and 'acceptance'. Conclusions: The study results can support animal health stakeholders in the development of antimicrobial stewardship programmes to promote appropriate antimicrobial use and limit the emergence of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Aims: This report documents the exposure of passengers and crew of a commercial international flight to the zoonotic pathogen Brucella canis after an infected dog aborted in the passenger cabin of the aircraft. This case demonstrates the challenges associated with brucellosis screening and the risks that airline personnel, airport employees and travellers face when animals with unrecognized zoonotic infections are transported. Methods/Results: The public health investigation of this case was conducted by the Centers for Disease Control, the Illinois Department of Health and the Illinois Department of Agriculture, in collaboration with a local veterinary clinic and several academic and federal diagnostic laboratories. It included an extensive diagnostic evaluation of the dam and aborted foetuses to confirm a diagnosis of canine brucellosis. Passengers, airline personnel and staff from the veterinary clinic where the dogs were treated underwent risk assessments, and clinic staff also received detailed guidance regarding infection prevention practices. Conclusions: Animal shelters and breeding programs are recommended to screen dogs routinely for brucellosis, but it is not unusual for domestic or imported animals to have unknown health histories, including the dog's brucellosis status, at the time of purchase, adoption, or re‐homing. Testing recommendations and requirements vary by state, making it challenging for state public health and animal health agencies to monitor and respond appropriately. This case highlights the importance of Brucella spp. screening in sexually intact dogs prior to breeding, purchase, or domestic or international transportation of the dogs. The transportation of pregnant dogs may present a previously unrecognized public health threat in addition to contributing to unnecessary stress and health risks for pregnant animals. [ABSTRACT FROM AUTHOR]