A RETROSPECTIVE STUDY OF DYSTOCIA IN EASTERN INDIGO SNAKES (DRYMARCHON COUPERI).

Medical and husbandry records of eastern indigo snakes (EIS) (Drymarchon couperi) housed at the Orianne Center for Indigo Conservation (OCIC) were reviewed to determine risk factors for developing dystocia. Thirty-four cases of dystocia were identified out of 104 successful breeding events between 2010 and 2020. The number of breeding events, age, body weight, housing, and selected blood parameters were reviewed for the female EIS successfully bred at OCIC between 2010 and 2020. Categorical data were evaluated with chi-square and the Fisher exact test, and the continuous data were evaluated using analysis of variance. Differences in data were considered statistically significant when P < 0.05. There was a significant difference seen between EIS that developed dystocia (EISd) and EIS that did not (EISn) for breeding events, with the virgin breeding event having more EISd than any other breeding event. Risk analysis of the data revealed that EIS were 9.28 times more likely to develop dystocia during the virgin breeding event than on subsequent breeding events. Age and housing were not significantly different between EISd and EISn for each breeding event, but body weight was, as EIS weighing less than 1.5 kg were found to be 4.43 times more likely to develop dystocia. There was also a significant difference between EISd and EISn in plasma sodium, calcium, ionized calcium, glucose, and albumin. When compared to EISn, EISd had lower plasma sodium, calcium, ionized calcium, and albumin and higher plasma glucose. There were also significant differences in the white blood cell differential count, as EISd had lower basophils and monocytes when compared to EISn. The majority of EISd were managed surgically, and retained eggs were more likely to be located within the right oviduct. This retrospective study is the first to evaluate potential causes of dystocia in EIS.

Predicting progression to diabetes in islet autoantibody positive children.

While full oral glucose tolerance test (OGTT) helps improve prediction, it requires intravenous access with 6 sample collections for glucose and C-peptide. The objective of this study was to explore less costly and less time-consuming options. All children being prospectively followed by the Diabetes Autoimmunity Study in the Young (DAISY) who had a complete baseline OGTT and at least one confirmed islet autoantibody (Ab+) were included in this study (n = 68). Of 68 Ab+ subjects with a baseline OGTT, 25 developed diabetes after a mean follow-up 5.7 yrs, at a mean age of 12.4 yrs. Univariate proportional hazards (PH) models suggested that age at seroconversion, number of Ab+, IA-2A levels, HbA1c and metabolic variables from the OGTT predicted progression to diabetes, while HLA DR3/4, BMI, levels of IAA or GADA did not. Five multivariate PH predictive models were similar (p = 0.32). All five models included age at seroconversion, number of Ab+, IA-2A levels and HbA1c, and in addition included: model 1 - 1 h glucose and 1 h C-peptide; model 2 - 2 h glucose and 2 h C-peptide; model 3 - glucose sum and C-peptide sum; model 4 - glucose AUC and C-peptide AUC; and model 5: index 60. A model containing age at seroconversion, number of Ab+, IA-2A levels, HbA1c, 1 h glucose and 1 h C-peptide was as predictive for type 1 diabetes progression as models including all sum or AUC values for glucose and C-peptide from full OGTT. The performance of this model should be confirmed in an independent population of Ab+ children.

Short-Stalked Prosthecomicrobium hirschii Cells Have a Caulobacter-Like Cell Cycle.

UNLABELLED: The dimorphic alphaproteobacterium Prosthecomicrobium hirschii has both short-stalked and long-stalked morphotypes. Notably, these morphologies do not arise from transitions in a cell cycle. Instead, the maternal cell morphology is typically reproduced in daughter cells, which results in microcolonies of a single cell type. In this work, we further characterized the short-stalked cells and found that these cells have a Caulobacter-like life cycle in which cell division leads to the generation of two morphologically distinct daughter cells. Using a microfluidic device and total internal reflection fluorescence (TIRF) microscopy, we observed that motile short-stalked cells attach to a surface by means of a polar adhesin. Cells attached at their poles elongate and ultimately release motile daughter cells. Robust biofilm growth occurs in the microfluidic device, enabling the collection of synchronous motile cells and downstream analysis of cell growth and attachment. Analysis of a draft P. hirschii genome sequence indicates the presence of CtrA-dependent cell cycle regulation. This characterization of P. hirschii will enable future studies on the mechanisms underlying complex morphologies and polymorphic cell cycles. IMPORTANCE: Bacterial cell shape plays a critical role in regulating important behaviors, such as attachment to surfaces, motility, predation, and cellular differentiation; however, most studies on these behaviors focus on bacteria with relatively simple morphologies, such as rods and spheres. Notably, complex morphologies abound throughout the bacteria, with striking examples, such as P. hirschii, found within the stalked Alphaproteobacteria. P. hirschii is an outstanding candidate for studies of complex morphology generation and polymorphic cell cycles. Here, the cell cycle and genome of P. hirschii are characterized. This work sets the stage for future studies of the impact of complex cell shapes on bacterial behaviors.

Epigenetic Treatment of Neuropsychiatric Disorders: Autism and Schizophrenia.

Neuropsychiatric disorders are a heterogeneous group of conditions that often share underlying mitochondrial dysfunction and biological pathways implicated in their pathogenesis, progression, and treatment. To date, these disorders have proven notoriously resistant to molecular-targeted therapies, and clinical options are relegated to interventional types, which do not address the core symptoms of the disease. In this review, we discuss emerging epigenetic-driven approaches using novel acylcarnitine esters (carnitinoids) that act on master regulators of antioxidant and cytoprotective genes and mitophagic pathways. These carnitinoids are actively transported, mitochondria-localizing, biomimetic coenzyme A surrogates of short-chain fatty acids, which inhibit histone deacetylase and may reinvigorate synaptic plasticity and protect against neuronal damage. We outline these neuroprotective effects in the context of treatment of neuropsychiatric disorders such as autism spectrum disorder and schizophrenia.

Timescales and Frequencies of Reversible and Irreversible Adhesion Events of Single Bacterial Cells.

In the environment, most bacteria form surface-attached cell communities called biofilms. The attachment of single cells to surfaces involves an initial reversible stage typically mediated by surface structures such as flagella and pili, followed by a permanent adhesion stage usually mediated by polysaccharide adhesives. Here, we determine the absolute and relative timescales and frequencies of reversible and irreversible adhesion of single cells of the bacterium Caulobacter crescentus to a glass surface in a microfluidic device. We used fluorescence microscopy of C. crescentus expressing green fluorescent protein to track the swimming behavior of individual cells prior to adhesion, monitor the cell at the surface, and determine whether the cell reversibly or irreversibly adhered to the surface. A fluorescently labeled lectin that binds specifically to polar polysaccharides, termed holdfast, discriminated irreversible adhesion events from reversible adhesion events where no holdfast formed. In wild-type cells, the holdfast production time for irreversible adhesion events initiated by surface contact (23 s) was 30-times faster than the holdfast production time that occurs through developmental regulation (13 min). Irreversible adhesion events in wild-type cells (3.3 events/min) are 15-times more frequent than in pilus-minus mutant cells (0.2 events/min), indicating the pili are critical structures in the transition from reversible to irreversible surface-stimulated adhesion. In reversible adhesion events, the dwell time of cells at the surface before departing was the same for wild-type cells (12 s) and pilus-minus mutant cells (13 s), suggesting the pili do not play a significant role in reversible adhesion. Moreover, reversible adhesion events in wild-type cells (6.8 events/min) occur twice as frequently as irreversible adhesion events (3.3 events/min), demonstrating that most cells contact the surface multiple times before transitioning from reversible to irreversible adhesion.

Cryptosporidium serpentis Surveillance in Free-Ranging Snakes to Inform a Reintroduction Strategy for the Eastern Indigo Snake (Drymarchon couperi).

Understanding risk factors associated with reintroductions is important for making informed decisions within an adaptive framework. Biosecurity measures minimizing the risk of the introduction or spread of transmissible diseases are a priority when considering the release of captive-reared wildlife. Eastern indigo snake (EIS; Drymarchon couperi) reintroductions have been occurring in Alabama since 2010 and in Florida since 2017. During this effort the pathogen Cryptosporidium serpentis was detected, affecting several of the captive breeding snakes. Infected snakes were quarantined and removed from breeding efforts, which reduced snakes available for the reintroduction projects. To make informed management decisions about future reintroduction strategies, 155 free-ranging snakes were sampled at the two release sites and a third site in Georgia to evaluate the natural occurrence of C. serpentis. Additionally, 72 free-ranging EIS and other species incidentally encountered throughout the EIS range were tested opportunistically. All snakes sampled at the three focal sites tested negative, but one opportunistically tested EIS from South Florida tested positive. These results indicate that C. serpentis is present in the environment in at least one location, but at low levels. Our results suggest that, pending additional surveillance, C. serpentispositive snakes should not be included in reintroduction efforts, and that maintaining a high level of biosecurity is important in captive breeding programs.

Notch signaling drives intestinal graft-versus-host disease in mice and nonhuman primates.

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4ß7 in conventional T cells while preserving α4ß7 in regulatory T cells, with findings suggesting increased ß1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4ß7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

Microfluidic device for automated synchronization of bacterial cells.

We report the development of an automated microfluidic "baby machine" to synchronize the bacterium Caulobacter crescentus on-chip and to move the synchronized populations downstream for analysis. The microfluidic device is fabricated from three layers of poly(dimethylsiloxane) and has integrated pumps and valves to control the movement of cells and media. This synchronization method decreases incubation time and media consumption and improves synchrony quality compared to the conventional plate-release technique. Synchronized populations are collected from the device at intervals as short as 10 min and at any time over four days. Flow cytometry and fluorescence cell tracking are used to determine synchrony quality, and cell populations synchronized in minimal growth medium with 0.2% glucose (M2G) and peptone yeast extract (PYE) medium contain >70% and >80% swarmer cells, respectively. Our on-chip method overcomes limitations with conventional physical separation methods that consume large volumes of media, require manual manipulations, have lengthy incubation times, are limited to one collection, and lack precise temporal control of collection times.

Spatiotemporal single-cell profiling reveals that invasive and tissue-resident memory donor CD8+ T cells drive gastrointestinal acute graft-versus-host disease.

Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8+ T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8+ T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (TRM) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3 and CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8+ T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8+ TRM cells during aGVHD in primate transplant recipients.

Gluten intake and risk of thyroid peroxidase autoantibodies in the Diabetes Autoimmunity Study In the Young (DAISY).

PURPOSE: Autoimmune diseases co-occur, perhaps due to common risk factors. The age at gluten introduction and gluten intake in early childhood has been associated with the autoimmunity preceding celiac disease (CD) and type-1 diabetes (T1D). We explored their associations with the development of thyroid autoimmunity. METHODS: DAISY has prospectively followed children at increased risk for T1D and CD since 1993. During follow-up, 107 children developed thyroid autoimmunity, defined as positivity for autoantibodies against thyroid peroxidase on at least two study visits. Age at gluten introduction was ascertained from food history interviews every 3 months until 15 months of age. Gluten intake (g/day) at age 1-2 years was estimated using a food frequency questionnaire. RESULTS: From multivariable Cox regression, there was no association between the age of gluten introduction nor the amount of gluten intake and development of thyroid autoimmunity. However, females (hazard ratio = 2.19, 95% CI: 1.46, 3.27) and cases of islet autoimmunity (HR = 2.20, 95% CI: 1.39, 3.50) were significantly more likely to develop thyroid autoimmunity, while exposure to environmental tobacco smoke decreased the risk (HR = 0.46, 95% CI: 0.30, 0.71). CONCLUSIONS: Neither the age of gluten introduction nor the amount of gluten consumed in early childhood is associated with risk of thyroid autoimmunity.

Isolation of a Highly Purified HSC-enriched CD34+CD90+CD45RA- Cell Subset for Allogeneic Transplantation in the Nonhuman Primate Large-animal Model.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a common treatment for patients suffering from different hematological disorders. Allo-HCT in combination with hematopoietic stem cell (HSC) gene therapy is considered a promising treatment option for millions of patients with HIV+ and acute myeloid leukemia. Most currently available HSC gene therapy approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly progenitor cells and only very few HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side effects in nontarget cells. We have previously shown that purified CD34+CD90+CD45RA- cells are enriched for multipotent HSCs with long-term multilineage engraftment potential, which can reconstitute the entire hematopoietic system in an autologous nonhuman primate transplant model. Here, we tested the feasibility of transplantation with purified CD34+CD90+CD45RA- cells in the allogeneic setting in a nonhuman primate model. METHODS: To evaluate the feasibility of this approach, CD34+CD90+CD45RA- cells from 2 fully major histocompatibility complex-matched, full sibling rhesus macaques were sort-purified, quality controlled, and transplanted. Engraftment and donor chimerism were evaluated in the peripheral blood and bone marrow of both animals. RESULTS: Despite limited survival due to infectious complications, we show that the large-scale sort-purification and transplantation of CD34+CD90+CD45RA- cells is technically feasible and leads to rapid engraftment of cells in bone marrow in the allogeneic setting and absence of cotransferred T cells. CONCLUSIONS: We show that purification of an HSC-enriched CD34+ subset can serve as a potential stem cell source for allo-HCTs. Most importantly, the combination of allo-HCT and HSC gene therapy has the potential to treat a wide array of hematologic and nonhematologic disorders.

Continuous Glucose Monitoring Predicts Progression to Diabetes in Autoantibody Positive Children.

CONTEXT: Accurate measures are needed for the prediction and diagnosis of type 1 diabetes (T1D) in at-risk persons. OBJECTIVE: The purpose of this study was to explore the value of continuous glucose monitoring (CGM) in predicting T1D onset. DESIGN AND SETTING: The Diabetes Autoimmunity Study in the Young (DAISY) prospectively follows children at increased risk for development of islet autoantibodies (islet autoantibody positive; Ab+) and T1D. PARTICIPANTS: We analyzed 23 Ab+ participants with available longitudinal CGM data. MAIN OUTCOME MEASURE: CGM metrics as glycemic predictors of progression to T1D. RESULTS: Of 23 Ab+ participants with a baseline CGM, 8 progressed to diabetes at a median age of 13.8 years during a median follow-up of 17.7 years (interquartile range, 14.6 to 22.0 years). Compared with nonprogressors, participants who progressed to diabetes had significantly increased baseline glycemic variability (SD, 29 vs 21 mg/dL; P = 0.047), daytime sensor average (122 vs 106 mg/dL; P = 0.02), and daytime sensor area under the curve (AUC, 470,370 vs 415,465; P = 0.047). They spent 24% of time at >140 mg/dL and 12% at >160 mg/dL compared with, respectively, 8% and 3% for nonprogressors (both P = 0.005). A receiver-operating characteristic curve analysis showed an AUC of 0.85 for percentage of time spent at >140 or 160 mg/dL. The cutoff of 18% time spent at >140 mg/dL had 75% sensitivity, 100% specificity, and a 100% positive predictive value for diabetes prediction, although these values could change because some nonprogressors may develop diabetes with longer follow-up. CONCLUSIONS: Eighteen percent or greater CGM time spent at >140 mg/dL predicts progression to diabetes in Ab+ children.

Serum-based inhibition of pitviper venom by eastern indigo snakes (Drymarchon couperi).

When organisms possess chemical defenses, their predators may eventually evolve resistance to their toxins. Eastern indigo snakes (Drymarchon couperi; EIS) prey on pitvipers and are suspected to possess physiological resistance to their venom. In this study, we formally investigated this hypothesis using microassays that measured the ability of EIS blood sera to inhibit (A) hemolytic and (B) snake venom metalloproteinase (SVMP) activity of copperhead (Agkistrodon contortrix) venom. To serve as controls, we also tested the inhibitory ability of sera from house mice (Mus musculus) and checkered gartersnakes (Thamnophis marcianus), a snake that does not feed on pitvipers. Sera from both EIS and gartersnakes inhibited over 60% of SVMP activity, while only EIS sera also inhibited venom hemolytic activity (78%). Our results demonstrate that EIS serum is indeed capable of inhibiting two of the primary classes of toxins found in copperhead venom, providing the first empirical evidence suggesting that EIS possess physiological resistance to venom upon injection. Because we documented resistance to hemolytic components of pitviper venom within EIS but not gartersnakes, we speculate this resistance may be driven by selection from feeding on pitvipers while resistance to SVMP may be relatively widespread among snakes.

Patterns of head shape and scutellation in Drymarchon couperi (Squamata: Colubridae) reveal a single species.

Krysko et al. (2016a) used analyses of DNA sequence data to reveal two genetic lineages of Drymarchon couperi. The Atlantic lineage contained specimens from southeastern Georgia and eastern peninsular Florida, and the Gulf Coast lineage contained specimens from western and southern peninsular Florida as well as western Florida, southern Alabama, and southern Mississippi. In a second paper Krysko et al. (2016b) analyzed morphological variation of the two lineages, which allowed them to restrict D. couperi to the Atlantic lineage and to describe the Gulf Coast lineage as a new species, Drymarchon kolpobasileus. This taxonomic discovery was remarkable for such a large, wide-ranging species and was notable for its impact on conservation. Because of population declines, particularly in western Florida, southern Alabama, and southern Mississippi, D. couperi (sensu lato) was listed as Threatened under the Endangered Species Act (United States Fish and Wildlife Service 1978, 2008) and repatriation of the species to areas where it had been extirpated was listed as a priority conservation goal (United States Fish and Wildlife Service 1982, 2008). Such repatriation efforts were attempted in Alabama, Florida, Georgia, and South Carolina, starting in 1977 (Speake et al. 1987), but failed to create viable populations, likely because too few snakes were released at too many sites (Guyer et al. 2019; Folt et al. 2019a). A second attempt at repatriation was started in 2010 and concentrated on release of snakes at a single site in Alabama (Stiles et al. 2013). However, Krysko et al. (2016a) criticized this repatriation effort because it appeared to involve release of D. couperi (sensu stricto) into the geographic region occupied by D. kolpobasileus (as diagnosed in Krysko et al. 2016b).

Taxonomic and conservation implications of population genetic admixture, mito-nuclear discordance, and male-biased dispersal of a large endangered snake, Drymarchon couperi.

Accurate species delimitation and description are necessary to guide effective conservation of imperiled species, and this synergy is maximized when multiple data sources are used to delimit species. We illustrate this point by examining Drymarchon couperi (Eastern Indigo Snake), a large, federally-protected species in North America that was recently divided into two species based on gene sequence data from three loci and heuristic morphological assessment. Here, we re-evaluate the two-species hypothesis for D. couperi by evaluating both population genetic and gene sequence data. Our analyses of 14 microsatellite markers revealed 6-8 genetic population clusters with significant admixture, particularly across the contact zone between the two hypothesized species. Phylogenetic analyses of gene sequence data with maximum-likelihood methods suggested discordance between mitochondrial and nuclear markers and provided phylogenetic support for one species rather than two. For these reasons, we place Drymarchon kolpobasileus into synonymy with D. couperi. We suggest inconsistent patterns between mitochondrial and nuclear DNA are driven by high dispersal of males relative to females. We advocate for species delimitation exercises that evaluate admixture and gene flow in addition to phylogenetic analyses, particularly when the latter reveal monophyletic lineages. This is particularly important for taxa, such as squamates, that exhibit strong sex-biased dispersal. Problems associated with over-delimitation of species richness can become particularly acute for threatened and endangered species, because of high costs to conservation when taxonomy demands protection of more individual species than are supported by accumulating data.

Influence of channel position on sample confinement in two-dimensional planar microfluidic devices.

We report enhanced sample confinement on microfluidic devices using a combination of electrokinetic flow from adjacent control channels and electric field shaping with an array of channels perpendicular to the sample stream. The basic device design consisted of a single first dimension (1D) channel, intersecting an array of 32 or 96 parallel second dimension (2D) channels. To minimize sample dispersion and leakage into the parallel channels as the sample traversed the sample transfer region, control channels were placed to the left and right of the 1D and waste channels. The electrokinetic flow from the control channels confined the sample stream and acted as a buffer between the sample stream and the 2D channels. To further enhance sample confinement, the electric field was shaped parallel to the sample stream by placing the channel array in close proximity to the sample transfer region. Using COMSOL Multiphysics, initial work focused on simulating the electric fields and fluid flows in various device geometries, and the results guided device design. Following the design phase, we fabricated devices with 40, 80, and 120 microm wide control channels and evaluated the sample stream width as a function of the electric field strength ratio in the control and 1D channels (E(C)/E(1D)). For the 32 channel design, the 40 and 80 microm wide control channels produced the most effective sample confinement with stream widths as narrow as 75 microm, and for the 96 channel design, all three control channel widths generated comparable sample stream widths. Comparison of the 32 and 96 channel designs showed sample confinement scaled easily with the length of the sample transfer region.

Dietary glycemic index, development of islet autoimmunity, and subsequent progression to type 1 diabetes in young children.

CONTEXT: Dietary factors may trigger or exacerbate the autoimmune disease process. OBJECTIVE: Our objective was to examine dietary glycemic index (GI) and glycemic load (GL) for association with islet autoimmunity (IA) development, and progression from IA to type 1 diabetes. DESIGN: The Diabetes Autoimmunity Study in the Young follows children at increased genetic type 1 diabetes risk. Diet is collected prospectively via a parent-reported food frequency questionnaire. SETTING: This was an observational study of children in the Denver area. PATIENTS: A total of 1776 Diabetes Autoimmunity Study in the Young children younger than 11.5 yr was included in the study. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: IA, defined as the presence of autoantibodies to insulin, glutamic acid decarboxylase, or protein tyrosine phosphatase at two consecutive visits, or the presence of autoantibodies at one visit and diabetic on the next consecutive visit was determined. Type 1 diabetes was diagnosed by a physician. A total of 89 subjects developed IA, and 17 subsequently developed type 1 diabetes during follow-up. Our hypothesis was formulated after data collection. RESULTS: GI and GL were not associated with IA development. More rapid progression to type 1 diabetes in children with IA was associated with higher dietary GI (hazard ratio: 2.20; 95% confidence interval: 1.17-4.15) and marginally associated with GL (hazard ratio: 1.59; 95% confidence interval: 0.96-2.64) at the first IA-positive visit. CONCLUSIONS: Higher dietary GI and GL are not associated with IA development, but higher GI is associated with more rapid progression to type 1 diabetes in children with IA, perhaps due to increased demand on the beta-cell to release insulin. Further study is needed to confirm this finding and identify the underlying biological mechanism.

Water-assisted femtosecond laser machining of electrospray nozzles on glass microfluidic devices.

Using water-assisted femtosecond laser machining, we fabricated electrospray nozzles on glass coverslips and on assembled microfluidic devices. Machining the nozzles after device assembly facilitated alignment of the nozzles over the microchannels. The basic nozzle design is a through-hole in the coverslip to pass liquids and a trough machined around the through-hole to confine the electrospray and prevent liquid from wicking across the glass surface. Electrospray from the nozzles was stable with and without pressure-driven flow applied and was evaluated using mass spectra of the peptide bradykinin.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

Omega-3 polyunsaturated fatty acid intake and islet autoimmunity in children at increased risk for type 1 diabetes.

CONTEXT: Cod liver oil supplements in infancy have been associated with a decreased risk of type 1 diabetes mellitus in a retrospective study. OBJECTIVE: To examine whether intakes of omega-3 and omega-6 fatty acids are associated with the development of islet autoimmunity (IA) in children. DESIGN, SETTING, AND PARTICIPANTS: A longitudinal, observational study, the Diabetes Autoimmunity Study in the Young (DAISY), conducted in Denver, Colorado, between January 1994 and November 2006, of 1770 children at increased risk for type 1 diabetes, defined as either possession of a high diabetes risk HLA genotype or having a sibling or parent with type 1 diabetes. The mean age at follow-up was 6.2 years. Islet autoimmunity was assessed in association with reported dietary intake of polyunsaturated fatty acids starting at age 1 year. A case-cohort study (N = 244) was also conducted in which risk of IA by polyunsaturated fatty acid content of erythrocyte membranes (as a percentage of total lipids) was examined. MAIN OUTCOME MEASURE: Risk of IA, defined as being positive for insulin, glutamic acid decarboxylase, or insulinoma-associated antigen-2 autoantibodies on 2 consecutive visits and still autoantibody positive or having diabetes at last follow-up visit. RESULTS: Fifty-eight children developed IA. Adjusting for HLA genotype, family history of type 1 diabetes, caloric intake, and omega-6 fatty acid intake, omega-3 fatty acid intake was inversely associated with risk of IA (hazard ratio [HR], 0.45; 95% confidence interval [CI], 0.21-0.96; P = .04). The association was strengthened when the definition of the outcome was limited to those positive for 2 or more autoantibodies (HR, 0.23; 95% CI, 0.09-0.58; P = .002). In the case-cohort study, omega-3 fatty acid content of erythrocyte membranes was also inversely associated with IA risk (HR, 0.63; 95% CI, 0.41-0.96; P = .03). CONCLUSION: Dietary intake of omega-3 fatty acids is associated with reduced risk of IA in children at increased genetic risk for type 1 diabetes.