Patterns and trends in lake concentrations of dissolved organic carbon in a landscape recovering from environmental degradation and widespread acidification.

Concentrations of dissolved organic carbon (DOC) have increased in lakes throughout North America and Europe over the last three decades. Recovery from acid deposition and climate change have both been postulated as the primary mechanisms for the increase in DOC. To provide a clearer insight into the mechanisms responsible for increasing DOC we evaluated changes in lake and peat porewater chemistry collected in an area of approximately 33,000 km2 surrounding Sudbury, Ontario, a region undergoing dramatic recovery from acidic deposition. DOC concentrations varied considerably among the 44 lakes and over time (samples annually from 1981 to 2018), but the Sens Slope value showed a strong increase in lake DOC concentration over time, at 0.05 mg/ L y-1 (p < 0.001) that was related to increasing pH [0.03 units y-1, p < 0.001] and decreasing lake SO4 concentration [-0.24 mg/ L y-1; p < 0.001], but showed no relationship with temperature or precipitation. Similar strong relationships between DOC and pH (positive) and SO4 (negative) were observed in 18 peatlands sampled in the region. In a spatial analysis of 82 lakes sampled in 2018, concentrations of DOC in lakes were highest in flatter catchments with a greater wetland area, suggesting that wetlands are a major source of DOC in lakes. Optical properties of DOC obtained from extracts of wetland and upland soils at 6 catchments could be distinguished, primarily due to upland litter extracts having distinct optical properties from mineral soils or wetland soils. Optical properties of DOC in lakes however were inconsistent with those measured in soil extracts indicating that they are not useful for distinguishing DOC sources in these lakes. A predictive model was developed to explain DOC trends within Sudbury lakes using a stepwise linear regression combined with hierarchical partitioning to confirm the most influential processes on DOC. Almost 50% of the variability in DOC change in the 44 lakes was explained by the magnitude in lake pH change, catchment size and catchment sparse tree cover showing that recovery from acidic deposition is overwhelmingly responsible for increasing DOC in Sudbury lakes.

Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches.

The epithelial lining of the small intestine consists of multiple cell types, including Paneth cells and goblet cells, that work in cohort to maintain gut health. 3D in vitro cultures of human primary epithelial cells, called organoids, have become a key model to study the functions of Paneth cells and goblet cells in normal and diseased conditions. Advances in these models include the ability to skew differentiation to particular lineages, providing a useful tool to study cell type specific function/dysfunction in the context of the epithelium. Here, we use comprehensive profiling of mRNA, microRNA and long non-coding RNA expression to confirm that Paneth cell and goblet cell enrichment of murine small intestinal organoids (enteroids) establishes a physiologically accurate model. We employ network analysis to infer the regulatory landscape altered by skewing differentiation, and using knowledge of cell type specific markers, we predict key regulators of cell type specific functions: Cebpa, Jun, Nr1d1 and Rxra specific to Paneth cells, Gfi1b and Myc specific for goblet cells and Ets1, Nr3c1 and Vdr shared between them. Links identified between these regulators and cellular phenotypes of inflammatory bowel disease (IBD) suggest that global regulatory rewiring during or after differentiation of Paneth cells and goblet cells could contribute to IBD aetiology. Future application of cell type enriched enteroids combined with the presented computational workflow can be used to disentangle multifactorial mechanisms of these cell types and propose regulators whose pharmacological targeting could be advantageous in treating IBD patients with Crohn's disease or ulcerative colitis.

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner.

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi-a process termed 'cell shedding'. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes.

Non-viral immune electrogene therapy induces potent antitumour responses and has a curative effect in murine colon adenocarcinoma and melanoma cancer models.

Antitumour efficacy of electroporated pEEV, coding for granulocyte-macrophage colony-stimulating factor and the B7-1 costimulatory immune molecule (pEEVGmCSF-b7.1) in growing solid tumours, was investigated and compared with a standard plasmid. Application of pEEVGmCSF-b7.1 led to complete tumour regression in 66% of CT26-treated tumours and 100% in the B16F10-treated tumours at day 150 post-treatment. pEEVGmCSF-b7.1 treatment was found to significantly enhance levels of both innate and adaptive immune populations in tumour and systemic sites, which corresponded to significantly increased tissue levels of proinflammatory cytokines including interferon-γ (IFN-γ) and interleukin-12 (IL-12). In contrast, pEEVGmCSF-b7.1 treatment significantly reduced the T-regulatory populations and also the anti-inflammatory cytokine IL-10. Upon further characterisation of functional immune responses, we observed a significant increase in cytotoxic (CD107a+) and IFN-γ-producing natural killer cells and also significantly more in IL-12-producing B cells. Importantly, splenocytes isolated from pEEVGmCSF-b7.1-treated 'cured' mice were tumour-specific and afforded significant protection in a tumour rechallenge model (Winn assay). Our data indicate that electroimmunogene therapy with the non-viral pEEVGmCSF-b7.1 is able to induce potent and durable antitumour immune responses that significantly reduce primary and also secondary tumour growth, and thus represents a solid therapeutic platform for pursuing future clinical trials.

Enhancement of electroporation facilitated immunogene therapy via T-reg depletion.

Regulatory T cells (T-regs) can negatively impact tumor antigen-specific immune responses after infiltration into tumor tissue. However, depletion of T-regs can facilitate enhanced anti-tumor responses, thus augmenting the potential for immunotherapies. Here we focus on treating a highly aggressive form of cancer using a murine melanoma model with a poor prognosis. We utilize a combination of T-reg depletion and immunotherapy plasmid DNA delivered into the B16F10 melanoma tumor model via electroporation. Plasmids encoding murine granulocyte macrophage colony-stimulating factor and human B71 were transfected with electroporation into the tumor and transient elimination of T-regs was achieved with CD25-depleting antibodies (PC61). The combinational treatment effectively depleted T-regs compared to the untreated tumor and significantly reduced lung metastases. The combination treatment was not effective in increasing the survival, but only effective in suppression of metastases. These results indicate the potential for combining T-reg depletion with immunotherapy-based gene electrotransfer to decrease systemic metastasis and potentially enhance survival.

Natural killer cells protect mice from DSS-induced colitis by regulating neutrophil function via the NKG2A receptor.

Natural killer (NK) cells are traditionally considered in the context of tumor surveillance and infection defense but their role in chronic inflammatory disorders such as inflammatory bowel disease is less clear. Here, we investigated the role of NK cells in dextran sodium sulfate (DSS)-induced colitis in mice. Depletion of NK cells impairs the survival of mice with colitis and is linked with dramatic increases in colonic damage, leukocyte infiltration, and pro-inflammatory profiles. Mice depleted of NK cells had increased numbers of neutrophils in colons and mesenteric lymph nodes, compared with control mice, in addition to acquiring a hyper-activation status. In vitro and in vivo studies demonstrate that NK cells downregulate pro-inflammatory functions of activated neutrophils, including reactive oxygen species and cytokine production, by direct cell-to-cell contact involving the NK cell-inhibitory receptor NKG2A. Our results indicate an immunoregulatory mechanism of action of NK cells attenuating DSS-induced colitis neutrophil-mediated inflammation and tissue injury via NKG2A-dependent mechanisms.

Use of bioluminescence imaging to track neutrophil migration and its inhibition in experimental colitis.

Inflammatory bowel disease (IBD) is associated with neutrophil infiltration into the mucosa and crypt abscesses. The chemokine interleukin (IL)-8 [murine homologues (KC) and macrophage inflammatory protein (MIP)-2] and its receptor CXCR2 are required for neutrophil recruitment; thus, blocking this engagement is a potential therapeutic strategy. In the present study, we developed a preclinical model of neutrophil migration suitable for investigating the biology of and testing new drugs that target neutrophil trafficking. Peritoneal exudate neutrophils from transgenic ß-actin-luciferase mice were isolated 12h after intraperitoneal injection with thioglycollate, and were assessed phenotypically and functionally. Exudate cells were injected intravenously into recipients with dextran sodium sulphate (DSS)-induced colitis followed by bioluminescence imaging of whole-body and ex vivo organs at 2, 4 and 16-22h post-transfer. Anti-KC antibody or an isotype control were administered at 20 µg/mouse 1h before transfer, followed by whole-body and organ imaging 4h post-transfer. The peritoneal exudate consisted of 80% neutrophils, 39% of which were CXCR2(+) . In vitro migration towards KC was inhibited by anti-KC. Ex vivo bioluminescent imaging showed that neutrophil trafficking into the colon of DSS recipients was inhibited by anti-KC 4h post-cell transfer. In conclusion, this study describes a new approach for investigating neutrophil trafficking that can be used in preclinical studies to evaluate potential inhibitors of neutrophil recruitment.

Verifying the stroke-free phenotype by structured telephone interview.

BACKGROUND AND PURPOSE: Case-control, cohort, and twin studies support a genetic contribution to ischemic stroke risk. Sibling pair linkage methods require identification of concordant or discordant siblings or both. We designed and tested a structured telephone interview to verify the stroke-free phenotype. METHODS: A coordinator unaware of medical record data used an 8-item questionnaire to conduct a structured telephone interview of 70 outpatients aged >60 years. The questionnaire inquired about the sudden onset of deficits in strength, sensation, vision, and language. A subject was defined as stroke free by interview if responses to all items on the questionnaire were negative. Results of the telephone interview were compared with data obtained from a systematic medical record review (benchmark). RESULTS: Interview time was 5 minutes or less for all subjects. All subjects who began the interview completed it. Records were reviewed in all subjects. Medical record review detected ischemic stroke or transient ischemic attack (TIA), or both, in 5 patients (7%). There were no significant differences in sex distribution or risk factor rates in patients who were designated stroke free or not stroke free by interview. Having 1 or more positive items on the questionnaire was significantly associated with finding stroke (P<0.001), TIA (P<0.001), or either stroke or TIA (P<0.001), on medical record review. The telephone interview had a sensitivity of 1.0 (95% CI 0.48 to 1.0), specificity of 0.86 (95% CI 0.75 to 0.93), positive predictive value of 0.36 (95% CI 0.13 to 0.65), and negative predictive value of 1.0 (95% CI 0.94 to 1.0). CONCLUSIONS: Our instrument can identify the stroke-free individual with a high degree of confidence in a very efficient manner. It may be particularly suited for centralized verification of stroke discordancy in multicentered sib-pair genetic studies.

Recognition of facial emotional expressions from moving and static displays by individuals with mental retardation.

Moving and static videotaped and photographic displays of posed emotional expressions were presented to 12 adults with mental retardation and 12 without mental retardation to investigate the role of movement in the recognition of facial expressions of emotion. Participants chose the corresponding emotion portrayed by the displays from among six written and pictorial labels of the emotions. Results indicated that individuals with mental retardation were significantly poorer at identifying anger, fear, disgust, and surprise. Both groups performed significantly better on the moving as opposed to the static videotaped displays of the emotions sad and angry. Visual-perceptual limitations are likely contributors to the poorer performance of the group with mental retardation in recognizing moving and static facial expressions of emotion.

Gender difference in jaw pain induced by clenching.

Seven normal male and seven normal female volunteers performed three types of biting exercises: an intermittent contraction at maximum voluntary bite force (MVBF) to pain intolerance; a ramp intermittent contraction starting at 10% MVBF and increasing 10% every 10 s; and a sustained biting at 100% MVBF to pain intolerance. The following measurements were made on the first and second days before exercise: a pre- and post-exertional overall jaw pain level; maximum pain-free jaw opening; bilateral masseter pressure pain threshold (PPT) and intolerance (PPI). The results showed that only females presented an increased overall pain level on the second day and a significant decrease in pain-free jaw opening, but no significant decrease of PPT and PPI. These results suggest that females respond differently than males to exertional jaw pain, by increasing their pain response 24 h later.

TOWARDS A THEORY OF QUARK AND LEPTON MASSES.

Has any progress been made on understanding and predicting the 13 parameters which describe the observed masses and mixing angles of the quarks and leptons? Arguments are given in favor of pursuing schemes in which grand unified and family symmetries provide many relations among these 13 parameters. A sequence of simple assumptions leads to a supersymmetric SO(10) theory with 8 predictions: tan ß, mt, Vcb, ms, ms/md, mu/md, Vub and the amount of CP violation J. These predictions are presented, together with experiments which will test them.

Clearance of recombinant tissue factor pathway inhibitor (TFPI) in rabbits.

The pharmacokinetics of recombinant tissue factor pathway inhibitor (TFPI) after an intravenous bolus injection was studied in rabbits. Clearance of TFPI was followed by measurement of the radioactivity of the 125I-labelled compound in the whole plasma or the trichloroacetic acid precipitate and by quantitation of the functional TFPI activity of the unlabelled compound using a tissue factor-induced coagulation assay. When iodinated TFPI was used, the ratios of the trichloroacetic acid precipitable counts vs. that of the whole plasma was about 1 in the first 10 min after TFPI injection, but this ratio gradually decreased to less than 0.5 after 2 h. This result suggested that the iodinated TFPI in the plasma was partially degraded after prolonged circulation in the animal. When unlabelled TFPI was used, the clearance of TFPI activity from the plasma exhibited bi-exponential elimination kinetics with a rapid alpha phase half-life (t1/2 alpha) of 2.3 min, and a terminal beta phase half-life (t1/2 beta) of 79 min. The plasma clearance was 4.2 ml kg-1 min-1. The tissue distribution of intravenously administered 125I-TFPI in the rabbit was studied using whole-body autoradiography. At 3 min after dosing, significant levels of TFPI were apparent in the liver, kidney, and other highly blood perfused tissues. Significant levels of 125I-TFPI-derived radioactivity were also apparent in the liver and kidney at 30 min after intravenous administration. The localization within the liver demonstrated a mottled appearance, suggesting regions of higher uptake within the liver. In the kidney, the outer cortex consistently revealed the highest activity.

Functional analysis of the rat insulin-like growth factor I gene and identification of an IGF-I gene promoter.

Insulin-like growth factor I (IGF-I) mediates many of the systemic growth-promoting effects of growth hormone and also functions as a locally acting growth stimulator. In mammals, IGF-I gene expression is complicated, as the gene is transcribed and processed into multiple mRNAs (ranging in length from less than 1 to nearly 7.5 kb) that encode at least two protein precursors. As a step toward understanding the regulation of IGF-I, we report the complete organization of the rat IGF-I gene, including identification of the structural determinants for all IGF-I mRNA species, and an initial functional analysis of its promoters. The gene is composed of 6 exons distributed over nearly 80 kb of chromosomal DNA and is structurally heterogeneous. Several transcription start sites were identified within IGF-I exons 1 and 2, adjacent to presumptive promoters 1 and 2, respectively, and at least three polyadenylation sites were mapped to exon 6. To test promoter function, fusion genes were constructed linking fragments of IGF-I DNA to a reporter plasmid. Chimeric genes containing at least 395 bp of DNA from the 5'-flanking region of exon 1 enhanced luciferase activity after transfection into the IGF-I-producing SK-N-MC cell line, while fusion plasmids containing up to 1,300 bp of DNA from the 5'-flanking region of exon 2 were inactive. Relative levels of IGF-I mRNAs containing exons 1 or 2 varied among different rat tissues, although in response to acute or chronic growth hormone treatment both classes of transcripts were induced coordinately in rat liver. These observations represent the first thorough characterization of a mammalian IGF-I gene, and provide a starting point for defining the mechanisms by which growth hormone and other trophic factors regulate IGF-I gene expression.

Preschool siblings of handicapped children: interactions with mothers, brothers, and sisters.

The purpose of this investigation was to examine similarities and differences between young (aged 3 years to 6 years 9 months) siblings of handicapped and nonhandicapped children in their behavioral interactions with their mothers, brothers, and sisters. Behavior of mothers toward the different groups of children also was examined. Results revealed few differences between sibling groups in the quantity or quality of their interactions with family members. In comparison to the matched control children, siblings of handicapped children engaged in more parallel play and social play, and were more nurturing but no more likely to interact aggressively or to be commanding or directive with their brothers or sisters. Mothers in the experimental group were found to target significantly more nurturant behaviors toward their children compared to control mothers and were significantly more likely to deliver commands, directives, and reprimands to siblings of handicapped children than to any other child. Results are discussed in terms of their correspondence to previous observational and interview research.

Evolution of insulin-like growth factor II: characterization of the mouse IGF-II gene and identification of two pseudo-exons.

We have cloned the mouse insulin-like growth factor II (IGF-II) gene as a series of overlapping cosmid and lambda recombinants and have characterized its six exons. The gene extends over approximately 12 kb of mouse chromosome 7 and is located 18 kb 3' to the insulin 2 gene and in the same transcriptional polarity. Exons 1-3 encode distinct 5' untranslated regions and are transcribed by three different promoters, P1, P2, and P3, into three IGF-II mRNAs sharing common coding and 3' untranslated sequences. Promoters P2 and P3 each contain a TATA box and appear to direct transcription from single initiation sites. By contrast, exon 1 has three major transcriptional start sites distributed over 556 nucleotides, and P1 lacks a TATA region and other typical transcriptional control sequences. Exons 4-6 code for the 180-amino-acid IGF-II precursor, and exon 6 also contains a 3,045-nucleotide 3' untranslated region which ends at a single polyadenylation site. In addition to six functional IGF-II exons, we identified two 5' "pseudo-exons," which appear to be evolutionarily retained remnants of an alternative promoter-exon cassette that is active in human IGF-II. Loss of the homolog of this promoter, which directs "adult-specific" expression of the IGF-II gene in some human tissues, may explain the disappearance of this growth factor from most murine tissues in the early postnatal period.

T-tube with tragus cartilage flange in long-term middle ear ventilation.

This was a study of the use of tragus cartilage as a flange for a T-tube in the hope of achieving long-term middle ear ventilation. This procedure was performed on 31 ears in 25 patients. Average length of follow-up was 24.2 months, ranging from 6 to 39 months. Five patients had tympanomastoid procedures done in conjunction with the cartilage tube. One of the patients with a tympanomastoid procedure had rejection of the T-tube and cartilage at 11 months postoperatively. None of the tubes without another procedure was rejected.

Gastroduodenal artery-duodenal fistula: a complication of continuous floxuridine (FUDR) infusion into the gastroduodenal artery.

This case report describes a patient who for 31 months has received regional intrahepatic chemotherapy from a continuous infusion pump and who developed a gastroduodenal artery-duodenal fistula, a previously unreported complication of regional infusion therapy. The patient presented with signs and symptoms of upper gastrointestinal bleeding. The clinical evaluation and management are described. An angiogram was performed through the auxillary septum to identify the source of bleeding. The possible etiologic factors in this case are discussed. We believe that this complication will continue to be rare, but health care providers should be aware of its presentation and its preferred method of evaluation and management.

Comparison of two non-disconnect CAPD delivery systems.

Intraluminal transmission of bacteria remains a significant factor in the morbidity and procedural success of CAPD. A worsening rate of peritonitis in a longstanding CAPD program (first patients 1978) led to a search for a system which might allow a lesser rate of peritonitis. Simplicity in the procedure was a requirement. For this reason disconnect systems were not considered. The Travenol spike system was prospectively compared with Delmed leur lock in terms of rate of peritonitis and difficulty in training. The former had been used by the center since inception of the CAPD program. 28 patients new to CAPD were alternately assigned to each system without other bias, including diagnosis, age, sex, or race. The study, while ongoing, was analyzed at 12 months. 66 patient months were involved with each system. The peritonitis rates were: 2.2 episodes: patient year with the Travenol; 0.9 episode:patient year for the Delmed systems. The Delmed system appears to provide a lower rate of peritonitis. In addition, less manual dexterity, steadiness, and hand eye coordination are necessary.

Psychosocial characteristics of preschool siblings of handicapped and nonhandicapped children.

This project examined psychosocial characteristics of 24 preschool-aged siblings of handicapped children in relation to a control group of 22 siblings of nonhandicapped children. Subjects were matched on family size and income, sibling age, birth order, sex, age spacing, and marital status of their parents. Results indicated no statistically significant differences between groups of children on measures of perceived self-competence and acceptance, understanding of developmental disabilities, empathy, and child care responsibility. Significant group differences were found where brothers of handicapped children were rated by their mothers as being more depressed and aggressive than brothers of nonhandicapped control children. Sisters of handicapped children were rated by mothers as being more aggressive than sisters of nonhandicapped children. Sisters of handicapped children and brothers of nonhandicapped children had significantly fewer privileges and more restrictions on their home activities than other groups. Results are discussed in relation to previous research on older siblings of handicapped children and the general literature on family stress and childhood disability and disease. The importance of examining sibling functioning via multiple measures of child behavior is stressed.