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Background: Patients with diabetes at risk of food insecurity face cost barriers to healthy eating and, as a result, poor health outcomes. Population health management strategies are needed to improve food security in real-world health system settings. We seek to test the effect of a prescription produce program, 'Eat Well' on cardiometabolic health and healthcare utilization. We will also assess the implementation of an automated, affirmative outreach strategy., Methods: We will recruit approximately 2400 patients from an integrated academic health system in the southeastern United States as part of a two-arm parallel hybrid type 1 pragmatic randomized controlled trial. Patients with diabetes, at risk for food insecurity, and a recent hemoglobin A1c reading will be eligible to participate. The intervention arm receives, 'Eat Well', which provides a debit card with $80 (added monthly) for 12 months valid for fresh, frozen, or canned fruits and vegetables across grocery retailers. The control arm does not. Both arms receive educational resources with diabetes nutrition and self-management materials, and information on existing care management resources. Using an intent-to-treat analysis, primary outcomes include hemoglobin A1C levels and emergency department visits in the 12 months following enrollment. Reach and fidelity data will be collected to assess implementation., Discussion: Addressing food insecurity, particularly among those at heightened cardiometabolic risk, is critical to equitable and effective population health management. Pragmatic trials provide important insights into the effectiveness and implementation of 'Eat Well' and approaches like it in real-world settings., Registration: ClinicalTrials.gov Identifier: NCT05896644; Clinical Trial Registration Date: 2023-06-09., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Published by Elsevier Inc.)

Surveillance data from wildlife and poultry was used to describe the spread of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b in British Columbia (B.C.) and the Yukon, Canada from September 2022 - June 2023 compared to the first "wave" of the outbreak in this region, which occurred April - August 2022, after the initial viral introduction. Although the number of HPAI-positive poultry farms and wildlife samples was greater in "Wave 2", cases were more tightly clustered in southwestern B.C. and the most commonly affected species differed, likely due to an influx of overwintering waterfowl in the area. Eight HPAI genetic clusters, representing seven genotypes and two inter-continental viral incursions, were detected, with significant variation in the relative abundance of each cluster between the waves. Phylogenetic data suggests multiple spillover events from wild birds to poultry and mammals but could not rule out transmission among farms and among mammals.

High intra-specific genetic diversity is associated with adaptive potential, which is key for resilience to global change. However, high variation may also support deleterious alleles through genetic load, thereby increasing the risk of inbreeding depression if population sizes decrease. Purging of deleterious variation has been demonstrated in some threatened species. However, less is known about the costs of declines and inbreeding in species with large population sizes and high genetic diversity even though this encompasses many species globally that are expected to undergo population declines. Caribou is a species of ecological and cultural significance in North America with a wide distribution supporting extensive phenotypic variation but with some populations undergoing significant declines resulting in their at-risk status in Canada. We assessed intra-specific genetic variation, adaptive divergence, inbreeding, and genetic load across populations with different demographic histories using an annotated chromosome-scale reference genome and 66 whole-genome sequences. We found high genetic diversity and nine phylogenomic lineages across the continent with adaptive diversification of genes, but also high genetic load among lineages. We found highly divergent levels of inbreeding across individuals, including the loss of alleles by drift but not increased purging in inbred individuals, which had more homozygous deleterious alleles. We also found comparable frequencies of homozygous deleterious alleles between lineages regardless of nucleotide diversity. Thus, further inbreeding may need to be mitigated through conservation efforts. Our results highlight the "double-edged sword" of genetic diversity that may be representative of other species atrisk affected by anthropogenic activities., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

The emergence of ophidiomycosis (or snake fungal disease) in snakes has prompted increased awareness of the potential effects of fungal infections on wild reptile populations. Yet, aside from Ophidiomyces ophidiicola , little is known about other mycoses affecting wild reptiles. The closely related genus Paranannizziopsis has been associated with dermatomycosis in snakes and tuataras in captive collections, and P. australasiensis was recently identified as the cause of skin infections in non-native wild panther chameleons ( Furcifer pardalis ) in Florida, USA. Here we describe five cases of Paranannizziopsis spp. associated with skin lesions in wild snakes in North America and one additional case from a captive snake from Connecticut, USA. In addition to demonstrating that wild Nearctic snakes can serve as a host for these fungi, we also provide evidence that the genus Paranannizziopsis is widespread in wild snakes, with cases being identified in Louisiana (USA), Minnesota (USA), Virginia (USA), and British Columbia (Canada). Phylogenetic analyses conducted on multiple loci of the fungal strains we isolated identified P. australasiensis in Louisiana and Virginia; the remaining strains from Minnesota and British Columbia did not cluster with any of the described species of Paranannizziopsis , although the strains from British Columbia appear to represent a single lineage. Finally, we designed a pan- Paranannizziopsis real-time PCR assay targeting the internal transcribed spacer region 2. This assay successfully detected DNA of all described species of Paranannizziopsis and the two potentially novel taxa isolated in this study and did not cross-react with closely related fungi or other fungi commonly found on the skin of snakes. The assay was 100% sensitive and specific when screening clinical (skin tissue or skin swab) samples, although full determination of the assay's performance will require additional follow up due to the small number of clinical samples ( n = 14 from 11 snakes) available for testing in our study. Nonetheless, the PCR assay can provide an important tool in further investigating the prevalence, distribution, and host range of Paranannizziopsis spp. and facilitate more rapid diagnosis of Paranannizziopsis spp. infections that are otherwise difficult to differentiate from other dermatomycoses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lorch, Winzeler, Lankton, Raverty, Snyman, Schwantje, Thacker, Knowles, Cai and Grear.)

Monodispersed polyethylene glycol diacrylate (PEGDA)/acrylic acid (AA) microgels with a tuneable negative charge and macroporous internal structure have been produced using a Lego-inspired droplet microfluidic device. The surface charge of microgels was controlled by changing the content of AA in the monomer mixture from zero (for noncharged PEGDA beads) to 4 wt%. The macroporosity of the polymer matrix was introduced by adding 20 wt% of 600-MW polyethylene glycol (PEG) as a porogen material into the monomer mixture. The porogen was successfully leached out with acetone after UV-crosslinking, which resulted in micron-sized cylindrical pores with crater-like morphology, uniformly arranged on the microgel surface. Negatively charged PEGDA/AA beads showed improved adsorption capacity towards positively charged organic dyes (methylene blue and rhodamine B) compared to neutral PEGDA beads and high repulsion of negatively charged dye molecules (methyl orange and congo red). Macroporous microgels showed better adsorption properties than nonporous beads, with a maximum adsorption capacity towards methylene blue of 45 mg/g for macroporous PEGDA/AA microgels at pH 8.6, as compared to 23 mg/g for nonporous PEGDA/AA microgels at the same pH. More than 98% of Cu(II) ions were removed from 50 ppm solution at pH 6.7 using 2.7 mg/mL of macroporous PEGDA/AA microgel. The adsorption of cationic species was significantly improved when pH was increased from 3 to 9 due to a higher degree of ionization of AA monomeric units in the polymer network. The synthesized copolymer beads can be used in drug delivery to achieve improved loading capacity of positively charged therapeutic agents and in tissue engineering, where a negative charge of scaffolds coupled with porous structure can help to achieve improved permeability of high-molecular-weight metabolites and nutrients, and anti-fouling activity against negatively charged species.

Introduction: Oncotype Dx (ODX) is a genetic assay that analyzes tumor recurrence risk and provides chemotherapy recommendations for T1-T2 stage, hormone receptor-positive, human epidermal growth factor receptor-negative, and nodal-negative breast cancer patients. Despite its established validity, the utilization of this assay is suboptimal. The study aims to evaluate factors that are associated with adherence rate with the testing guidelines and examine changes in utilization trends., Methods: This is a retrospective study, utilizing data from the National Cancer Database from 2010 to 2017. Patients who met the ODX testing guidelines were first evaluated for testing adherence. Secondly, all patients who underwent ODX testing were assessed to evaluate the trend in ODX utilization., Results: A total of 429,648 patients met the criteria for ODX, and 43.4% of this population underwent testing. Advanced age, racial minorities, low-income status, well-differentiated tumor grade, uninsured status, and treatment at community cancer centers were associated with a decreased likelihood of receiving ODX in eligible patients. Additionally, a notable amount of testing was performed on patients who did not meet the ODX testing criteria. Among the 295,326 patients that underwent ODX testing, 16.6% of patients were node-positive and 1.8% had T3 or T4 stage tumors., Conclusions: A considerable number of patients who were eligible for ODX did not receive it, indicating potential barriers to care and disparities in breast cancer treatment. ODX usage has been expanded to broader patient populations, indicating more research is needed to validate the effectiveness of the assay in these patient groups., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Dedicated trauma intensive care units (ICUs) staffed by surgical intensivists lead to better patient outcomes. Increased length of stay (LOS) leads to worse outcomes. Little research has focused on the effect of dedicated trauma medical-surgical units or ICU/medicalsurgical systems. In 2018, our Level 1 trauma center transitioned from 3 non-dedicated levels of care (ICU/stepdown unit/medical-surgical) to 2 dedicated levels of care (ICU/medical-surgical). Our objective was to look at patient outcomes pre- and post-intervention., Methods: Retrospective analysis of trauma registry data was performed on patients (age ≥18) admitted to the trauma service at a Level 1 rural trauma center over 46-months. In the pre-intervention group, step down and medical-surgical patients were combined as "Non-ICU" for analysis. Standard statistical analysis was performed., Results: Analysis included 6103 patients. The group demographics were similar, except pre-intervention patients had higher ISS and fewer comorbidities. Emergency department LOS decreased from 30 versus 13.9% ( P < .0001) and 15.9 versus 5.8% ( P < .0001) for greater than 3 and 6 hours, respectively. Median LOS decreased for all patients ( P < .0001). Mortality dropped from 9.0 versus 5.5% ( P = .0009) for ICU and 1.7 versus 0.26% ( P = .0013) for non-ICU patients. Overall patient mortality was level at 3.7%. Inpatient complications dropped from 9.9 versus 8.5% ( P = .07). Unplanned ICU readmissions were unchanged ( P = .4169). For patients with 3+ comorbidities, overall LOS dropped by 2 days ( P < .0001) and home discharge increased from 42.8 versus 51% ( P < .0001)., Conclusion: Implementation of 2 levels of dedicated care has decreased ED and hospital LOS for all trauma patients without increasing mortality or complications. Patients with extensive comorbidities saw the most improvements.

Conservation breeding programs are increasingly used as recovery actions for wild animals; bringing founders into captivity to rear captive populations for future reintroduction into the wild. The International Union for the Conservation of Nature recommends that founders should come from genetically close populations and should have sufficient genetic diversity to avoid mating among relatives. Genomic data are highly informative for evaluating founders due to their high resolution and ability to capture adaptive divergence, yet, their application in that context remains limited. Woodland caribou are federally listed as a Species at Risk in Canada, with several populations facing extirpation, such as those in the Rocky Mountains of Alberta and British Columbia (BC). To prevent local extirpation, Jasper National Park (JNP) is proposing a conservation breeding program. We examined single nucleotide polymorphisms for 144 caribou from 11 populations encompassing a 200,000 2  km area surrounding JNP to provide information useful for identifying appropriate founders for this program. We found that this area likely hosts a caribou metapopulation historically characterized by high levels of gene flow, which indicates that multiple sources of founders would be appropriate for initiating a breeding program. However, population structure and adaptive divergence analyses indicate that JNP caribou are closest to populations in the BC Columbia range, which also have suitable genetic diversity for conservation breeding. We suggest that collaboration among jurisdictions would be beneficial to implement the program to promote recovery of JNP caribou and possibly other caribou populations in the surrounding area, which is strategically at the periphery of the distribution of this endangered species., Supplementary Information: The online version contains supplementary material available at 10.1007/s10592-023-01540-3., Competing Interests: Conflict of interestThe authors have no relevant financial or non-financial interests to disclose., (© The Author(s) 2023.)

Introduction: Over the past decade, small bowel obstructions (SBO) have been increasingly managed non-operatively. Prior studies have looked at outcomes based on admission to surgical services (SS) or medicine services (MS), but most are restricted to operative patients. This study evaluates the outcomes of non-operative patients specifically., Methods: A 12-year retrospective cohort study of patients ≥18-years-old admitted with SBO within one healthcare system was performed. Only non-operative patients were included. Clinicodemographic characteristics and admission details were extracted from the electronic medical record. Statistical analysis was performed using the student's t-test, chi-square, and multivariable regression., Results: A total of 3278 patients were included, of which 933(28.4%) patients were admitted to a SS. MS patients were older (57.7 vs 54.7 years, P < .001) and more likely to have diabetes (24.1 vs 20.2%, P = .015), CHF (5.7 vs 3.1%, P = .002), and AKI (29.8 vs 16.7%, P < .001). SS patients were more likely to have cancer (19.3 vs 13.7%, P < .001). Univariate analysis showed admission to SS decreased length of stay (3.4 vs 4.1 days, P < .001) and index admission mortality (0.1 vs 2.2%, P < .001). On multivariable analysis, admission to a SS decreased admission mortality (OR 0.056), 30-day mortality (OR 0.15), and 180-day mortality (OR 0.307). Similarly, 30-day readmissions (OR 0.683) and 180-day readmission (OR 0.54) were also significantly decreased. Length of stay was decreased by .6 days ( P < .001)., Discussion: In patients with non-operative SBO, admission to a surgical service decreased length of stay, mortality, and readmission. Further work should be completed evaluating how increased comorbidities affect long term outcomes. However, significantly decreased length of stay and mortality continue to support surgical services admitting SBO patients.

Corynosoma strumosum (Acanthocephala), a widespread parasite of pinnipeds, is reported in marine foraging North American mink (Neogale vison) and river otter (Lontra canadensis) on Vancouver Island, British Columbia. This is the first confirmed case of infection by C. strumosum in river otters on the west coast of North America and may be the first confirmed case of infection in wild North American mink; C. strumosum has previously been reported in river otters in Europe (Lutra lutra) and in farmed mink fed with marine fish. We also detected a case of acanthocephalan associated peritonitis in a juvenile mink. Furthermore, though infections with Corynosoma spp. are often assumed to be accidental in mustelids, some C. strumosum individuals found in mink showed signs of reproductive activity. These findings indicate that mink may be a competent definitive host and represent a reservoir in coastal habitats although further research is needed to confirm this. Investigating whether river otters may be competent hosts and determine the prevalence of infection in coastal populations would determine the potential implications of C. strumosum for coastal otters and minks. Our report indicates that mink and possibly river otter living in coastal areas are vulnerable to this previously unreported parasitic infection with mortality risk, at least in juvenile individuals., (Copyright © 2022 Elsevier B.V. All rights reserved.)

From February to May 2021, four nonmigratory rams from the Radium-Stoddart bighorn sheep (BHS; Ovis canadensis) herd in the Rocky Mountains of southeastern British Columbia, Canada, died from infection with the giant liver fluke, Fascioloides magna. Affected animals were emaciated, weak, and lethargic or were found dead. Gross lesions, histopathology, and parasite burdens were consistent with those reported in experimentally infected BHS, domestic sheep, and other aberrant hosts. Although BHS range does not typically overlap with fluke-contaminated aquatic habitats, the change in migratory behavior recently observed in some Radium-Stoddart rams may have exposed the affected animals to F. magna. We describe clinical signs and gross and histopathologic findings of hepatobiliary trematodiasis associated with F. magna in free-ranging BHS. From experimental data and our findings, giant liver fluke is pathogenic and is a threat to the conservation of the Radium-Stoddart BHS herd and other BHS herds in endemic F. magna regions., (© Wildlife Disease Association 2022.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many wild and domestic animal species. Farmed American mink ( Neovison vison ) are particularly susceptible to infection. Outbreaks of SARS-CoV-2 were detected in farmed mink on three mink farms in British Columbia (BC), Canada between December 2020 and May 2021. In BC, mink farm density and proximity to wildlife habitats increase transmission risks from infected farmed mink. The objective of this study is to investigate the risk of SARS-CoV-2 spreading to and from wildlife in the area surrounding infected mink farms in BC, Canada, as well as to compare the effectiveness of physical and camera trapping surveillance methodologies., Methods: A combination of physical and camera trapping was used on and around three BC mink farms with active SARS-CoV-2 infections between January 22, 2021, and July 10, 2021. Samples from trapped animals, including escaped farmed mink, were tested for SARS-CoV-2. Camera images from one mink farm were reviewed to determine species and proximity to the mink barn., Results: Seventy-one animals of nine species were captured and sampled. Three captured mink tested positive for SARS-CoV-2 by polymerase chain reaction and serology; the remaining samples were negative for SARS-CoV-2. Genotyping of the three positive mink indicated these were domestic (vs. wild) mink. A total of 440 animals of 16 species were photographed at the one farm where cameras were deployed., Conclusion: Detection of SARS-CoV-2 in escaped farmed mink is concerning and demonstrates the potential for transmission from farmed mink to wildlife, particularly given the observation of wildlife known to be susceptible to SARS-CoV-2 near infected mink farms. Combined use of physical and camera trapping contributed to the breadth of the results and is strongly recommended for future surveillance., Competing Interests: Competing interests: None., (Public Health Agency of Canada, 2022.)

Introduction: Small bowel obstruction (SBO) is a common admission diagnosis. Prior research has shown improved length of stay and time to operation for SBO patients on surgical services (SS) compared to medical services (MS). This study evaluates the impact of admitting service on readmission and mortality., Methods: A 12-year retrospective cohort study of patients ≥18 years old, admitted with SBO to either a MS or SS within one health care system was performed. Clinicodemographic characteristics and admission details were extracted and reviewed. Statistical analyses performed included the Student's t-test, chi-square, and multivariable regression., Results: The study included 7921 patients, of which 3862 (48.8%) were admitted to a SS. No significant clinicodemographic differences existed between the groups except SS patients were more likely to have cancer (23.3% vs 15.2%, P < .0001) and to be within a 30-day post-operative period (9.4% vs 1.8%, P < .0001). On multivariable analysis, admission to a SS was associated with a decreased admission mortality (OR .70), 30-day mortality (OR .42), and 180-day mortality (OR .42). 30-day readmissions (OR .54) and 180-day readmission (OR .43) were also significantly decreased for SS patients. In patients requiring a procedure during admission, there was significantly decreased admission mortality (OR .684), 30-day mortality (OR .470), 180-day mortality (OR .431), 30-day readmission (OR .63), and 180-day readmission (OR .50)., Conclusion: In patients with SBO, admission to a SS confers decreased odds of readmission and mortality compared to MS. Future studies are needed to understand the management decisions potentially underlying these differences. These findings may help better define admission pathways and improve outcomes.

Prediabetes affects one in three people and has a 10% annual conversion rate to type 2 diabetes without lifestyle or medical interventions. Management of glycemic health is essential to prevent progression to type 2 diabetes. However, there is currently no commercially-available and noninvasive method for monitoring glycemic health to aid in self-management of prediabetes. There is a critical need for innovative, practical strategies to improve monitoring and management of glycemic health. In this study, using a dataset of 25,000 simultaneous interstitial glucose and noninvasive wearable smartwatch measurements, we demonstrated the feasibility of using noninvasive and widely accessible methods, including smartwatches and food logs recorded over 10 days, to continuously detect personalized glucose deviations and to predict the exact interstitial glucose value in real time with up to 84% and 87% accuracy, respectively. We also establish methods for designing variables using data-driven and domain-driven methods from noninvasive wearables toward interstitial glucose prediction.

Introduction: Diabetes prevalence continues to grow and there remains a significant diagnostic gap in one-third of the US population that has pre-diabetes. Innovative, practical strategies to improve monitoring of glycemic health are desperately needed. In this proof-of-concept study, we explore the relationship between non-invasive wearables and glycemic metrics and demonstrate the feasibility of using non-invasive wearables to estimate glycemic metrics, including hemoglobin A1c (HbA1c) and glucose variability metrics., Research Design and Methods: We recorded over 25 000 measurements from a continuous glucose monitor (CGM) with simultaneous wrist-worn wearable (skin temperature, electrodermal activity, heart rate, and accelerometry sensors) data over 8-10 days in 16 participants with normal glycemic state and pre-diabetes (HbA1c 5.2-6.4). We used data from the wearable to develop machine learning models to predict HbA1c recorded on day 0 and glucose variability calculated from the CGM. We tested the accuracy of the HbA1c model on a retrospective, external validation cohort of 10 additional participants and compared results against CGM-based HbA1c estimation models., Results: A total of 250 days of data from 26 participants were collected. Out of the 27 models of glucose variability metrics that we developed using non-invasive wearables, 11 of the models achieved high accuracy (<10% mean average per cent error, MAPE). Our HbA1c estimation model using non-invasive wearables data achieved MAPE of 5.1% on an external validation cohort. The ranking of wearable sensor's importance in estimating HbA1c was skin temperature (33%), electrodermal activity (28%), accelerometry (25%), and heart rate (14%)., Conclusions: This study demonstrates the feasibility of using non-invasive wearables to estimate glucose variability metrics and HbA1c for glycemic monitoring and investigates the relationship between non-invasive wearables and the glycemic metrics of glucose variability and HbA1c. The methods used in this study can be used to inform future studies confirming the results of this proof-of-concept study., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Under-reporting of drug use in the perinatal period is well-documented, and significantly limits the reach of proactive intervention approaches. The Wayne Indirect Drug Use Screener (WIDUS) focuses on correlates of drug use rather than use itself. This trial tested a computer-delivered, brief intervention designed for use with indirect screen-positive cases, seeking to motivate reductions in drug use without presuming its presence., Methods: Randomized clinical trial with 500 WIDUS-positive postpartum women recruited between August 14, 2012 and November 19, 2014. Participants were randomly assigned to either a time control condition or a single-session, tailored, indirect brief intervention. The primary outcome was days of drug use over the 6-month follow-up period; secondary outcomes included urine and hair analyses results at 3- and 6-month follow-up. All outcomes were measured by blinded evaluators., Results: Of the 500 participants (252 intervention and 248 control), 36.1% of participants acknowledged drug use in the 3 months prior to pregnancy, but 89% tested positive at the 6-month follow-up. Participants rated the intervention as easy to use (4.9/5) and helpful (4.4/5). Analyses revealed no between-group differences in drug use (52% in the intervention group, vs. 53% among controls; OR 1.03). Exploratory analyses also showed that intervention effects were not moderated by baseline severity, WIDUS score, or readiness to change., Conclusions: The present trial showed no evidence of efficacy for an indirect, single-session, computer-delivered, brief intervention designed as a complement to indirect screening. More direct approaches that still do not presume active drug use may be possible and appropriate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The U.S. National Pain Strategy calls for increased population research on "high-impact chronic pain" (ie, longstanding pain that substantially limits participation in daily activities). Using data from the nationally-representative Health and Retirement Study (HRS), we investigated the prevalence of high-impact chronic pain in U.S. adults older than age 50 overall and within population subgroups. We also explored sociodemographic variation in pain-related disability within specific activity domains. Data are from a subsample of HRS respondents (n = 1,925) who were randomly selected for a supplementary pain module in 2010. Our outcome was operationalized as pain duration of ≥7 months and a disability rating of ≥7 (0-10 scale) in at least 1 domain: family/home, leisure, social activities, work, or basic activities. Overall, 8.2% (95% confidence interval = 6.7-10.1%) of adults older than age 50 met criteria for high-impact chronic pain. This proportion rose to 17.1% (95% confidence interval = 12.3-23.4%) among individuals in the lowest wealth quartile. Prevalence differences according to education, race/ethnicity, and age were not significant. Arthritis and depression were significantly associated with high-impact pain in multivariable analysis. Among adults with any chronic pain, African American and individuals in the lowest wealth quartile reported more pain-related disability across activity domains., Perspective: High-impact chronic pain is unequally distributed among midlife and older U.S. adults. Efforts to reduce the burden of disabling chronic pain should prioritize socioeconomically vulnerable groups, who may have the least access to multimodal pain treatment to improve function., (Copyright © 2017 American Pain Society. All rights reserved.)

Excitatory glutamatergic synaptic transmission is critically dependent on maintaining an optimal number of postsynaptic AMPA receptors (AMPARs) at each synapse of a given neuron. Here, we show that presynaptic activity, postsynaptic potential, voltage-gated calcium channels (VGCCs) and UNC-43, the C. elegans homolog of CaMKII, control synaptic strength by regulating motor-driven AMPAR transport. Genetic mutations in unc-43, or spatially and temporally restricted inactivation of UNC-43/CaMKII, revealed its essential roles in the transport of AMPARs from the cell body and in the insertion and removal of synaptic AMPARs. We found that an essential target of UNC-43/CaMKII is kinesin light chain and that mouse CaMKII rescued unc-43 mutants, suggesting conservation of function. Transient expression of UNC-43/CaMKII in adults rescued the transport defects, while optogenetic stimulation of select synapses revealed CaMKII's role in activity-dependent plasticity. Our results demonstrate unanticipated, fundamentally important roles for UNC-43/CaMKII in the regulation of synaptic strength., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An ammonium picket porphyrin that targets bacterial membranes has been prepared and shown to bind to phosphatidylglycerol (PG), a bacterial lipid, when the lipid was in solution, contained within synthetic membrane vesicles, or when in Gram-negative and Gram-positive bacterial membranes. The multifunctional receptor was designed to interact with both the phosphate anion portion and neutral glycerol portion of the lipid headgroup. The receptor's affinity and selectivity for binding to surfactant vesicles or lipid vesicles that contain PG within their membranes was directly measured using fluorescence correlation spectroscopy (FCS). FCS demonstrated that the picket porphyrin's binding pocket was complementary for the lipid headgroup, since simple Coulombic interactions alone did not induce binding. (1)H NMR and isothermal titration calorimetry (ITC) were used to determine the receptor's binding stoichiometry, receptor-lipid complex structure, binding constant, and associated thermodynamic properties of complexation in solution. The lipid-receptor binding motif in solution was shown to mirror the binding motif of membrane-bound PG and receptor. Cell lysis assays with E. coli (Gram-negative) and Bacillus thuringiensis (Gram-positive) probed with UV/Visible spectrophotometry indicated that the receptor was able to penetrate either bacterial cell wall and to bind to the bacterial inner membrane.

The strength of synaptic communication at central synapses depends on the number of ionotropic glutamate receptors, particularly the class gated by the agonist AMPA (AMPARs). Cornichon proteins, evolutionarily conserved endoplasmic reticulum cargo adaptors, modify the properties of vertebrate AMPARs when coexpressed in heterologous cells. However, the contribution of cornichons to behavior and in vivo nervous system function has yet to be determined. Here, we take a genetic approach to these questions by studying CNI-1--the sole cornichon homolog in C. elegans. cni-1 mutants hyperreverse, a phenotype associated with increased glutamatergic synaptic transmission. Consistent with this behavior, we find larger glutamate-gated currents in cni-1 mutants with a corresponding increase in AMPAR number. Furthermore, we observe opposite phenotypes in transgenic worms that overexpress CNI-1 or vertebrate homologs. In reconstitution studies, we provide support for an evolutionarily conserved role for cornichons in regulating the export of vertebrate and invertebrate AMPARs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This experiment was conducted to test a hypothesis that lactating dairy cows fed 35% brown midrib (BMR) corn silage and 25% alfalfa hay (dry matter (DM) basis) would consume more DM around peak lactation compared with those fed conventional corn silage (CS), resulting in longer peak milk production. Twenty-eight multiparous Holstein cows were used starting at the onset of lactation through 180 d in milk (DIM). Treatments were formulated to maintain a forage-to-concentrate ratio of 60:40, differing only in the CS hybrids used. Two dietary treatments were assessed in a completely randomized design: total mixed ration based on conventional CS (CCS) and total mixed ration based on BMR silage. Through peak lactation (1-60 DIM), DM intake was not different between dietary treatments, whereas DM intake post-peak lactation (61-180 DIM) tended to increase by feeding the BMR diet compared with the CCS diet (25.8 vs. 24.7 kg/d). Cows fed the BMR diet tended to lose less body weight through peak lactation compared with those fed the CCS diet (-0.22 vs. -0.52 kg/d). Although milk yield was not different between dietary treatments through peak lactation, milk yield post-peak lactation increased by feeding the BMR diet compared with the CCS diet (41.0 vs. 38.8 kg/d). Yield of 3.5% fat-corrected milk was similar between dietary treatments throughout the experiment (41.4 kg/d, on average), but milk fat concentration decreased by feeding the BMR diet compared with the CCS diet post-peak lactation (3.47 vs. 3.80%). Overall milk protein concentration was similar between dietary treatments throughout the experiment (2.96%, on average), whereas milk protein yield tended to be higher for the BMR diet post-peak lactation compared with the CCS diet (1.19 vs.1.13 kg/d). Feeding BMR silage with a high dietary concentration of alfalfa hay maintained more body weight, but did not affect milk production through peak lactation; however, cows fed the BMR diet post-peak lactation consumed more feed and maintained longer peak milk yield, leading to greater overall milk production and milk protein yield., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Caenorhabditis elegans ftn-1 and ftn-2, which encode the iron-storage protein ferritin, are transcriptionally inhibited during iron deficiency in intestine. Intestinal specific transcription is dependent on binding of ELT-2 to GATA binding sites in an iron-dependent enhancer (IDE) located in ftn-1 and ftn-2 promoters, but the mechanism for iron regulation is unknown. Here, we identify HIF-1 (hypoxia-inducible factor -1) as a negative regulator of ferritin transcription. HIF-1 binds to hypoxia-response elements (HREs) in the IDE in vitro and in vivo. Depletion of hif-1 by RNA interference blocks transcriptional inhibition of ftn-1 and ftn-2 reporters, and ftn-1 and ftn-2 mRNAs are not regulated in a hif-1 null strain during iron deficiency. An IDE is also present in smf-3 encoding a protein homologous to mammalian divalent metal transporter-1. Unlike the ftn-1 IDE, the smf-3 IDE is required for HIF-1-dependent transcriptional activation of smf-3 during iron deficiency. We show that hif-1 null worms grown under iron limiting conditions are developmentally delayed and that depletion of FTN-1 and FTN-2 rescues this phenotype. These data show that HIF-1 regulates intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage., Competing Interests: The authors have declared that no competing interests exist.

The GenPlex™ HID System (Applied Biosystems - AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250-500pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Sclerosing angiomatoid nodular transformation (SANT) is a benign, proliferative vascular lesion affecting the spleen. Few reports detailing the cross sectional and PET appearance of this lesion are available, and the lesion's behavior with ⁹⁹(m)Tc-sulfur colloid scintigraphy is previously unreported. Sclerosing nodular transformation of the spleen shows increased tracer accumulation on positron emission tomography, and a central scar-like appearance with an enhancing capsule and radiating septae on CT and MR studies that reflects the gross and histopathological features of the lesion may be visible. An understanding of this pathological finding may allow prospective recognition of the sclerosing nodular transformation of the spleen on cross sectional imaging studies.

Mutations in LRRK2 are thus far the most frequent known cause of autosomal dominant and idiopathic Parkinson's disease (PD) with prevalent mutations being found within the GTPase (R1441C/G) and kinase (G2019S) domains. Previous in vitro studies have revealed that R1441C and G2019S mutations are associated with increased kinase activity. To better understand LRRK2-linked PD pathogenesis in vivo, we have generated transgenic C. elegans overexpressing human LRRK2 wild type, R1441C and G2019S in dopaminergic (DA) neurons. Overexpression of these LRRK2 proteins causes age-dependent DA neurodegeneration, behavioral deficits, and locomotor dysfunction that are accompanied by a reduction of dopamine levels in vivo. In comparison, R1441C and G2019S mutants cause more severe phenotypes than the wild type protein. Interestingly, treatment with exogenous dopamine rescues the LRRK2-induced behavioral and locomotor phenotypes. In contrast, expression of the GTP binding defective mutant, K1347A, or knockout of the C. elegans LRRK2 homolog, LRK-1, prevents the LRRK2-induced neurodegeneration and behavioral abnormalities. Hence, our transgenic LRRK2 C. elegans models recapitulate key features of PD including progressive neurodegeneration, impairment of dopamine-dependent behavior and locomotor function, and reduction in dopamine levels. Furthermore, our findings provide strong support for the critical role of GTPase/kinase activity in LRRK2-linked pathologies. These invertebrate models will be useful for studying pathogenesis of PD and for development of potential therapeutics for the disease., ((c) 2010 Elsevier Inc. All rights reserved.)

Ferritin is a ubiquitous protein that sequesters iron and protects cells from iron toxicity. Caenorhabditis elegans express two ferritins, FTN-1 and FTN-2, which are transcriptionally regulated by iron. To identify the cis-acting sequences and proteins required for iron-dependent regulation of ftn-1 and ftn-2 expression, we generated transcriptional GFP reporters corresponding to 5 '-upstream sequences of the ftn-1 and ftn-2 genes. We identified a conserved 63-bp sequence, the iron-dependent element (IDE), that is required for iron-dependent regulation of a ftn-1 GFP reporter in intestine. The IDE contains two GATA-binding motifs and three octameric direct repeats. Site-directed mutagenesis of the GATA sequences, singly or in combination, reduces ftn-1 GFP reporter expression in the intestine. In vitro DNA mobility shift assays show that the intestine-specific GATA protein ELT-2 binds to both GATA sequences. Inhibition of ELT-2 function by RNA interference blocks ftn-1 GFP reporter expression in vivo. Insertion of the IDE into the promoter region of a heterologous reporter activates iron-dependent transcription in intestine. These data demonstrate that the activation of ftn-1 and ftn-2 transcription by iron requires ELT-2 and that the IDE functions as an iron-dependent enhancer in intestine.

Synaptojanin is a lipid phosphatase required to degrade phosphatidylinositol 4,5 bisphosphate (PIP(2)) at cell membranes during synaptic vesicle recycling. Synaptojanin mutants in C. elegans are severely uncoordinated and are depleted of synaptic vesicles, possibly because of accumulation of PIP(2). To identify proteins that act downstream of PIP(2) during endocytosis, we screened for suppressors of synaptojanin mutants in the nematode C. elegans. A class of uncoordinated mutants called "fainters" partially suppress the locomotory, vesicle depletion, and electrophysiological defects in synaptojanin mutants. These suppressor loci include the genes for the NCA ion channels, which are homologs of the vertebrate cation leak channel NALCN, and a novel gene called unc-80. We demonstrate that unc-80 encodes a novel, but highly conserved, neuronal protein required for the proper localization of the NCA-1 and NCA-2 ion channel subunits. These data suggest that activation of the NCA ion channel in synaptojanin mutants leads to defects in recycling of synaptic vesicles.

The advantages of single nucleotide polymorphism (SNP) typing in forensic genetics are well known and include a wider choice of high-throughput typing platforms, lower mutation rates, and improved analysis of degraded samples. However, if SNPs are to become a realistic supplement to current short tandem repeat (STR) typing methods, they must be shown to successfully and reliably analyse the challenging samples commonly encountered in casework situations. The European SNPforID consortium, supported by the EU GROWTH programme, has developed a multiplex of 52 SNPs for forensic analysis, with the amplification of all 52 loci in a single reaction followed by two single base extension (SBE) reactions which are detected with capillary electrophoresis. In order to validate this assay, a variety of DNA extracts were chosen to represent problems such as low copy number and degradation that are commonly seen in forensic casework. A total of 40 extracts were used in the study, each of which was sent to two of the five participating laboratories for typing in duplicate or triplicate. Laboratories were instructed to carry out their analyses as if they were dealing with normal casework samples. Results were reported back to the coordinating laboratory and compared with those obtained from traditional STR typing of the same extracts using Powerplex 16 (Promega). These results indicate that, although the ability to successfully type good quality, low copy number extracts is lower, the 52-plex SNP assay performed better than STR typing on degraded samples, and also on samples that were both degraded and of limited quantity, suggesting that SNP analysis can provide advantages over STR analysis in forensically relevant circumstances. However, there were also additional problems arising from contamination and primer quality issues and these are discussed.

Y chromosome haplotype data was collected for 155 Irish males residing in the Republic of Ireland. Eleven short tandem repeat (STR) markers: DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439 were analysed and the allele and haplotype frequencies calculated. This Irish data is presented here and was found to be less diverse when compared with the neighbouring UK population.

Genetic analysis of the nematode Caenorhabditis elegans reveals that all dpy-5 alleles are dominant suppressors of bli-4 blistering. Molecular cloning of dpy-5 establishes that it encodes a cuticle procollagen, defects in which are responsible for the short-body, dumpy phenotype. The null mutation, e907 removes the entire coding region, whereas the dpy-5 reference allele, e61, contains a nonsense substitution. RT-PCR analysis and a dpy-5::gfp fusion show that dpy-5 is expressed only in hypodermal cells at all post-embryonic life-cycle stages. Variable expression of dpy-5 in V lineage-derived seam cells suggests an alternative regulatory mechanism in these cells. The dpy-5 gene product contains an Arg-X-X-Arg cleavage motif that could be recognized by a proprotein convertase, such as BLI-4. Mutation of this site cause a dominant dumpy phenotype suggesting Dpy-5 procollagen requires processing for normal cuticle production.

The European Consortium "High-throughput analysis of single nucleotide polymorphisms for the forensic identification of persons--SNPforID", has performed a selection of candidate Y-chromosome single nucleotide polymorphisms (SNPs) for making inferences on the geographic origin of an unknown sample. From more than 200 SNPs compiled in the phylogenetic tree published by the Y-Chromosome Consortium, and looking at the population studies previously published, a package of 29 SNPs has been selected for the identification of major population haplogroups. A "Major Y-chromosome haplogroup typing kit" has been developed, which allows the multiplex amplification of all 29 SNPs in a single reaction. Allele genotyping was performed with a single base extension reaction (minisequencing) detected by CE. The validation of the multiplex was performed in a total of 1126 unrelated males distributed among 12 worldwide populations. The approach takes advantage of the specific geographic distribution of the Y-chromosome haplogroups and demonstrates the utility of binary polymorphisms to infer the origin of a male lineage.

Previously reported Y chromosome STR haplotype databases for three UK population groups, plus additionally analysed samples, have been scrutinised for the presence of non-standard (intermediate, null and duplicated) alleles. These alleles have been characterised by sequencing, some showing changes in the repeat structure, and the frequencies reported. Mutation rates for each of the 13 STRs have been calculated when analysis of father-son pairs has been possible. An example illustrating the use of non-standard alleles in a large family tree is outlined.

A collaborative study was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the performance of Y-chromosome binary polymorphism analysis in different European laboratories. Four blood samples were sent to the laboratories, to be analysed for 11 Y-chromosome single nucleotide polymorphisms (SNPs): SRY-1532, M40, M35, M213, M9, 92R7, M17, P25, M18, M153 and M167. All the labs were also asked to submit a population study including these markers. All participating laboratories reported the same results, indicating the reproducibility and robustness of Y-chromosome SNP typing. A total of 535 samples from six different European populations were also analysed. In Galicia (NW Spain) and Belgium, the most frequent haplogroup was R1b*(xR1b1,R1b3df). Haplogroup F*(xK) is one of the most frequent in Austria and Denmark, while the lowest frequency appear in Belgium. Haplogroup frequencies found in this collaborative study were compared with previously published European Y-chromosome haplogroup data.

Eleven Y chromosome short tandem repeat markers: DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439, have been typed in the three main UK population groups: Caucasians, Afro-Caribbeans and South Asians. Existing PCR reactions were adapted to incorporate DYS437, DYS438 and DYS439. The observed 11 loci haplotypes and the individual allele frequencies for each locus are presented. Distinct differences for most markers were observed between the population groups studied.

Low threshold-activated or T-type calcium channels are postulated to mediate a variety of bursting and rhythmic electrical firing events. However, T-type channels' exact physiological contributions have been difficult to assess because of their incompletely defined pharmacology and the difficulty in isolating T-type currents from more robust high threshold calcium currents. A current in C. elegans pharyngeal muscle displays the kinetic features of a T-type calcium channel and is absent in animals homozygous for mutations at the cca-1 locus (see accompanying paper). cca-1 is expressed in pharyngeal muscle and encodes a protein (CCA-1) with strong homology to the alpha1 subunits of vertebrate T-type channels. We show that CCA-1 plays a critical role at the pharyngeal neuromuscular junction, permitting the efficient initiation of action potentials in response to stimulation by the MC motor neuron. Loss of cca-1 function decreases the chance that excitatory input from MC will successfully trigger an action potential, and reduces the ability of an animal to take in food. Intracellular voltage recordings demonstrate that when wild-type cca-1 is absent, the depolarizing phase of the pharyngeal action potential tends to plateau or stall near -30 mV, the voltage at which the CCA-1 channel is likely to be activated. We conclude that the CCA-1 T-type calcium channel boosts the excitatory effect of synaptic input, allowing for reliable and rapid depolarization and contraction of the pharyngeal muscle. We also show that the pharyngeal muscle employs alternative strategies for initiating action potentials in certain cases of compromised MC motor neuron function.

A novel human cytosolic flavin reductase, Nr1, was recently described that contains FMN, FAD, and NADPH cofactors. Though the targets of the related NADPH-dependent flavoprotein reductases, cytochrome P450 reductase, methionine synthase reductase, and nitric oxide synthase, are known, the cellular function of Nr1 is not clear. To explore expression and regulation of Nr1, we cloned fre-1, the Caenorhabditis elegans ortholog of Nr1, and discovered that it is transcribed as a bicistronic pre-mRNA together with dcs-1, the ortholog of the recently described scavenger mRNA decapping enzyme. We used the novel substrate, 7meGpppBODIPY, to demonstrate that DCS-1 has low micromolar specificity for guanine ribonucleotides with the 7me modification, whereas trimethylated G substrates are poor competitors. Contrary to earlier classification, DCS-1 is not a pyrophosphatase but a distant member of the Hint branch of the histidine triad superfamily of nucleotide hydrolases and transferases. These observations are consistent with the hypothesis that DCS-1 homologs may function in the metabolism of capped oligonucleotides generated following exosome-dependent degradation of short-lived mRNA transcripts. We find that fre-1 and dcs-1 are coordinately expressed through worm development, are induced by heat shock, and have a nearly identical expression profile in human tissues. Furthermore, immunocytochemical analysis of the endogenous proteins in COS cells indicates that both are present in the nucleus and concentrated in a distinct perinuclear structure. Though no connection between these enzymes had been anticipated, our data and data from global expression and protein association studies suggest that the two enzymes jointly participate in responses to DNA damage, heat shock, and other stresses.

The Caenorhabditis elegans unc-2 gene encodes a voltage-gated calcium channel alpha1 subunit structurally related to mammalian dihydropyridine-insensitive high-threshold channels. In the present paper we describe the characterization of seven alleles of unc-2. Using an unc-2 promoter-tagged green fluorescent protein construct, we show that unc-2 is primarily expressed in motor neurons, several subsets of sensory neurons, and the HSN and VC neurons that control egg laying. Examination of behavioral phenotypes, including defecation, thrashing, and sensitivities to aldicarb and nicotine suggests that UNC-2 acts presynaptically to mediate both cholinergic and GABAergic neurotransmission. Sequence analysis of the unc-2 alleles shows that e55, ra605, ra606, ra609, and ra610 all are predicted to prematurely terminate and greatly reduce or eliminate unc-2 function. In contrast, the ra612 and ra614 alleles are missense mutations resulting in the substitution of highly conserved residues in the C terminus and the domain IVS4-IVS5 linker, respectively. Heterologous expression of a rat brain P/Q-type channel containing the ra612 mutation shows that the glycine to arginine substitution affects a variety of channel characteristics, including the voltage dependence of activation, steady-state inactivation, as well as channel kinetics. Overall, our findings suggest that UNC-2 plays a pivotal role in mediating a number of physiological processes in the nematode and also defines a number of critical residues important for calcium channel function in vivo.

Motivation: The complete genomes of a number of organisms have already been sequenced. However, the vast majority of annotated genes are derived by gene prediction methods. It is important to not only validate the predicted coding regions but also to identify genes that may have been missed by these programs., Methods: We searched the entire C.elegans genomic sequence database maintained by the Sanger Center using human c-Src sequence in a TBLASN search. We have confirmed one of the predicted regions by isolation of a cDNA and carried out a phylogenetic analysis of Src kinase family members in the worm, fly and several vertebrate species., Results: Our analysis identified a novel tyrosine kinase in the C.elegans genome that contains functional features typical of the Src family kinases that we have designated as Src-1. The open reading frame contains a conserved N-terminal myristoylation site and a tyrosine residue within the C-terminus that is crucial for regulating the activity of Src kinases. Our phylogenetic analysis of Src family members from C. elegans, Drosophila and other higher organisms revealed a relationship among Src kinases from C. elegans and Drosophila.

Objective: To investigate genetic susceptibility to recurrent fevers, generalized severe myalgia, and migratory erythema in an Israeli Arab child with no family history of similar disease., Methods: DNA sequencing of exons 1-6 of the TNFRSF1A gene (formerly TNFR1) was performed in the patient and his parents to determine the presence of the autosomal-dominant tumor necrosis factor receptor-associated periodic syndrome (TRAPS); informative markers spanning the TNFRSF1A locus were used to genotype all available members of the patient's family. The TNFRSF1A gene was subsequently screened in 69 healthy Arab controls and 96 Caucasian controls. Formal forensic paternity testing was performed on the child., Results: We found a de novo missense mutation in exon 3 of the TNFRSF1A gene, involving a novel C-->T transition encoding a Cys70Arg (C70R) variant, in the Israeli Arab patient. Eight of the common familial Mediterranean fever (FMF) gene MEFV mutations were excluded. This mutation was not present in the parents or siblings, or among the 69 healthy Arab controls. However, another TNFRSF1A variant, Pro46Lys (P46L), was present in 1 of the Arab controls., Conclusion: We have identified a TNFRSF1A mutation associated with periodic fever in an Arab patient, and a TNFRSF1A variant, which is variably pathogenic in Caucasians, in an Arab control. This is the first report of a de novo mutation in periodic fevers in general, and also of TRAPS in the Arab population. These findings demonstrate the need to include TRAPS in the differential diagnosis of recurrent fevers in this population.

Kex2/subtilisin-like proteinase activity is required for the production of the adult cuticle in the nematode Caenorhabditis elegans. Deletion of the carboxy termini of four of the bli-4/kpc-4 convertase isoforms results in blistering of the adult cuticle. The blisters vary in severity (expressivity) and are not evident in all individuals (reduced penetrance). We have isolated 13 bli-4/kpc-4 mutants that arrest development in late embryogenesis. Using a PCR-based heteroduplex technique, we have identified nucleotide changes responsible for eight of these lethal mutations. The lesions reside within the first 12 exons that are shared by all of the bli-4/kpc-4 gene products, with the majority of mutations clustered within the protease domain. This finding suggests that the protease domain represents a large mutable target. Among these mutations, allele h384 represents a molecular null mutant in which the catalytically essential serine residue (Ser415) is replaced by phenylalanine. Novel missense mutations that change the identity of amino acids evolutionary conserved in all kex2/subtilisin-convertases highlight critical residues essential for activity. We examined the functional activity of BLI-4/KPC-4 products expressed from several lethal mutants by testing their effect on the variable penetrance of blistering exhibited by the e937 allele. We found that the combination of a bli-4/kpc-4 lethal mutation in trans to the bli-4(e937) mutation was sufficient to cause severe blistering in heteroallelic progeny, even in the presence of a known dominant suppressor.

Significant advances have recently been made in our understanding of the mechanisms of activation of proteins that require processing. Often this involves endoproteolytic cleavage of precursor forms at basic residues, and is carried out by a group of serine endoproteinases, termed the proprotein convertases. In mammals, seven different convertases have been identified to date. These act in both the regulated secretory pathway for the processing of prohormones and proneuropeptides and in the constitutive secretory pathway, in which a variety of proproteins are activated endoproteolytically. The recently completed sequence of the nematode Caenorhabditis elegans genome affords a unique opportunity to examine the entire proprotein convertase family in a multicellular organism. Here we review the nature of the family, emphasising the structural features, characteristic of the four nematode genes, that supply all of the necessary functions unique to this group of serine endoproteinases. Studies of the C. elegans genes not only provide important information about the evaluation of this gene family but should help to illuminate the roles of these proteins in mammalian systems. BioEssays 22:545-553, 2000., (Copyright 2000 John Wiley & Sons, Inc.)

Comparative genomic analysis was used to investigate the gene structure of the bli-4 locus from two related Caenorhabditis species, C. elegans and C. briggsae. In C. elegans, bli-4 is a complex gene encoding a member of the kex2/subtilisin-like family of proprotein convertases. Genomic sequence comparisons coupled with RT-PCR analysis identified five additional coding exons that had not been identified previously using standard recombinant DNA techniques. The C. briggsae gene was able to rescue both viable blistered and developmentally arrested mutants of C. elegans bli-4, demonstrating functional conservation. In addition, deletion analysis of conserved sequences outside of coding regions, combined with phenotypic rescue experiments, identified regulatory elements that alter the expression of the bli-4 gene. These results demonstrate the utility of genomic sequence comparisons of homologous genes in related species as an effective tool with which to dissect the functional information of complex genes.

The mel-32 gene in the free living soil nematode Caenorhabditis elegans encodes a serine hydroxymethyltransferase (SHMT) isoform. Seventeen ethylmethanesulfonate (EMS)-induced mutant alleles of mel-32(SHMT) have been generated, each of which causes a recessive maternal effect lethal phenotype. Animals homozygous for the SHMT mutations have no observable mutant phenotype, but their offspring display an embryonic lethal phenotype. The Mel-32 phenotype has been rescued with a transgenic array containing only mel-32(SHMT) genomic DNA. Heteroduplex analysis of the 17 alleles allowed 14 of the mutations to be positioned to small regions. Subsequent sequence analysis has shown that 16 of the alleles alter highly conserved amino acids, while one allele introduces a stop codon that truncates two thirds of the predicted protein. mel-32(SHMT) has a 55-60% identity at the amino acid level with both isoforms of SHMT found in yeast and humans and a 50% identity with the Escherichia coli isoform. The C. elegans mel-32 mutation represents the first case where SHMT has been shown to be an essential gene.

Purpose: To determine the effect(s) on glucose control, insulin dose, and circulating insulin levels of the addition of a sulfonylurea (glipizide) to the treatment regimen of patients with insulin-requiring type 2 diabetes mellitus., Patients and Methods: Thirty seven patients with type 2 diabetes mellitus taking insulin for at least 1 year prior to study and treated with > or = 40 U of insulin per day were recruited for a randomized, double-blind, placebo-controlled, crossover trial. Patients were treated with 3 months of insulin + placebo (I + P) and 3 months of insulin + glipizide (I + G), with an intermediate 1 month washout period using insulin therapy alone. Adjustments were made initially to the maximum dose of glipizide (40 mg/day), followed by insulin dose adjustments. Twenty-nine of the 37 patients demonstrated a significant C-peptide response to Ensure and were selected for analysis., Results: The fasting plasma glucose in the I + G arm was 6.8 (121.8 mg/dl) vs. 8.7 mmol/L (156.0 mg/dl) in the I + P arm, P < 0.001. Mean plasma glucose over 24 hours was 9.8 (176.9 mg/dl) for I + G vs. 11.3 mmol/L (203.8 mg/dl) for I + P, P < 0.001. Glycated hemoglobin was significantly different (9.8 I + G vs. 11.4% I + P, P < 0.008). The total daily insulin dose required was significantly lower with I + G (69.1 vs. 87.3 U, P < 0.0005). However, there were no significant differences in free insulin levels., Conclusion: The addition of a sulfonylurea (glipizide) to insulin therapy in patients with insulin-requiring type 2 diabetes mellitus taking large doses of insulin results in a rapid and substantial improvement in glucose control despite a significant reduction in insulin dose. Therefore, this form of combination therapy should be considered for patients with the above characteristics whose diet and exercise programs are correct but whose response to insulin therapy is inadequate.

Objective: Insulin lispro is a rapid-acting analog of human insulin that can be used to target the postprandial rise in plasma glucose. We designed an open-label randomized crossover study of type 2 diabetic patients with secondary failure of sulfonylurea therapy to determine whether improvement of postprandial hyperglycemia would affect total daily glucose control., Research Design and Methods: Twenty-five type 2 diabetic patients who were poorly controlled on a maximum dose of sulfonylureas were studied in a university hospital clinical research center. In one arm of the study, patients continued therapy with maximum-dose sulfonylureas. In the other arm, patients used a combination therapy with insulin lispro before meals and sulfonylureas. After 4 months, patients were crossed over to the opposite arm. Fasting plasma glucose (FPG) and 1- and 2-h postprandial glucose (after a standardized meal), HbA1c, total, HDL, and LDL cholesterol, and triglyceride levels were measured at the end of each arm of the study., Results: Insulin lispro in combination with sulfonylurea therapy significantly reduced 2-h postprandial glucose concentrations compared with sulfonylureas alone, from 18.6 to 14.2 mmol/l (P < 0.0001), and incremental postprandial glucose area from 617.8 to 472.9 mmol.min.1-1 (P < 0.0007). FPG levels were decreased from 10.9 to 8.5 mmol/l (P < 0.0001), and HbA1c values were reduced form 9.0 to 7.1% (P < 0.0001). Total cholesterol was significantly decreased in the lispro arm from 5.44 to 5.10 mmol/l (P < 0.02). HDL cholesterol concentrations were increased in the lispro arm from 0.88 to 0.96 mmol/l (P < 0.01). The patients weighed significantly more after lispro therapy than after sulfonylureas alone, but the difference was small in absolute terms (sulfonylurea therapy alone, 90.6 kg; lispro therapy, 93.8 kg; P < 0.0001). Two episodes of hypoglycemia (glucose concentrations, < 2.8 mmol/l) were reported by the patients while using lispro., Conclusions: Previously, it has not been possible to address the effect of treatment of postprandial hyperglycemia specifically. We have now shown that the treatment of postprandial hyperglycemia with insulin lispro markedly improves overall glucose control and some lipid parameters in patients with type 2 diabetes.

Background and Objectives: Quinolone antibiotics are used widely for the treatment of gonorrhea, but resistant strains appeared in Sydney in 1984, treatment failure with high-dose regimens in 1991, and isolates with very high minimal inhibitory concentrations (MICs) (16 mg/l) in 1994., Goals: To examine the frequency, source, and characteristics of Quinolone-resistant Neisseria gonorrhoeae (QRNG) in Sydney from 1991 to 1995 and to compare these data with those obtained from 1984 to 1990., Study Design: The antibiotic sensitivity, auxotype-serovar class, and geographic source of QRNG isolated in Sydney from January 1, 1991 to June 30, 1995 were analyzed., Results: One hundred seven QRNG were isolated from 97 patients from 1991 to 1995. The number, proportion, and MICs of QRNG increased slowly in the first 4 years of the study and rapidly in the last 6 months. Most QRNG were isolated from travelers entering Sydney from Asia. Twenty-seven different auxotype-serovar classes were detected including 6 auxotype-serovar classes in 14 isolates with high-level quinolone resistance (MIC, 16 mg/l)., Conclusions: QRNG isolated in Sydney during the past decade originated in Asia as multiple gonococcal subtypes and increased substantially in numbers and levels of resistance in 1995.

Background and Objectives: Fluoroquinolones are widely used oral agents for treating Neisseria gonorrhoeae. Resistance to these agents is sporadic and usually at a low level. Two instances of high-dose ciprofloxacin regimens failing in the treatment of gonococcal infection, caused by strains with high-level quinolone resistance, are reported., Study Design: This is a case report., Conclusion: High-level resistance to quinolone antibiotics resulting in treatment failure was observed in two distinct gonococcal isolates from patients infected in the Philippines (ciprofloxacin; minimal inhibitory contribution = 16 mg/l). Continued monitoring of the quinolone sensitivity of Neisseria gonorrhoeae is appropriate and prudent.

Many secreted proteins are excised from inactive proproteins by cleavage at pairs of basic residues. Recent studies have identified several serine endoproteases that catalyze this cleavage in the secretory pathways of yeast and metazoans. These enzymes belong to the kex2/subtilisin-like family of proprotein convertases. In this paper we describe the molecular characterization of the bli-4 gene from Caenorhabditis elegans, which was shown previously by genetic analysis of lethal mutants to be essential for the normal development of this organism. Sequencing of cDNA and genomic clones has revealed that bli-4 encodes gene products related to the kex2/subtilisin-like family of proprotein convertases. Analysis of bli-4 cDNAs has predicted four protein products, which we have designated blisterases A, B, C, and D. These protein products share a common amino terminus, but differ at the carboxyl termini, and are most likely produced from alternatively spliced transcripts. We have determined the molecular lesions for three bli-4 alleles (h199, h1010, and q508) that result in developmental arrest during late embryogenesis. In each case, the molecular lesions are within exons common to all of the BLI-4 isoforms. The original defining allele of bli-4, e937, is completely viable yet exhibits blistering of the adult cuticle. Molecular analysis of this allele revealed a deletion that removes exon 13, which is unique to blisterase A. No RNA transcript corresponding to exon 13 is detectable in the blistered mutants. These findings suggest that blisterase A is required for the normal function of the adult cuticle.(ABSTRACT TRUNCATED AT 250 WORDS)