Impacts of hydraulic redistribution on grass-tree competition vs facilitation in a semi-arid savanna.

A long-standing ambition in ecosystem science has been to understand the relationship between ecosystem community composition, structure and function. Differential water use and hydraulic redistribution have been proposed as one mechanism that might allow for the coexistence of overstory woody plants and understory grasses. Here, we investigated how patterns of hydraulic redistribution influence overstory and understory ecophysiological function and how patterns vary across timescales of an individual precipitation event to an entire growing season. To this end, we linked measures of sap flux within lateral and tap roots, leaf-level photosynthesis, ecosystem-level carbon exchange and soil carbon dioxide efflux with local meteorology data. The hydraulic redistribution regime was characterized predominantly by hydraulic descent relative to hydraulic lift. We found only a competitive interaction between the overstory and understory, regardless of temporal time scale. Overstory trees used nearly all water lifted by the taproot to meet their own transpirational needs. Our work suggests that alleviating water stress is not the reason we find grasses growing in the understory of woody plants; rather, other stresses, such as excessive light and temperature, are being ameliorated. As such, both the two-layer model and stress gradient hypothesis need to be refined to account for this coexistence in drylands.

Blood Lead Levels in Young Children: US, 2009-2015.

OBJECTIVES: To evaluate trends in blood lead levels in children <6 years of age, this Quest Diagnostics Health Trends report builds on previously reported National Health and Nutrition Examination Survey data with a much larger national group and adds more detail and novel assessments. STUDY DESIGN: This report describes the results from a 6-year retrospective study (May 2009-April 2015) based on >5 million blood lead level results (including >3.8 million venous results) from children <6 years old living in all 50 states and the District of Columbia. We evaluated yearly changes and examined demographic categories including sex, pre-1950s housing construction, poverty income ratios (PIRs), Medicaid enrollment status, and geographic regions. RESULTS: Among children <6 years old, 3.0% exhibited blood lead levels ≥5.0 µg/dL (high). There were significant differences in high blood lead levels based on sex, pre-1950s housing construction quintiles, and PIR <1.25 and PIR >5 (all P < .01). Health and Human Services regions, states, and 3-digit ZIP code areas exhibited drastically different frequencies of high blood lead levels and blood lead levels ≥10.0 µg/dL (very high). Generally, levels declined over time for all groups. CONCLUSION: Examination of more than 5 million venous blood lead level results in children younger than 6 years old allowed for a robust, detailed analysis of blood lead level group results by geography and other criteria that are prohibited with the narrower National Health and Nutrition Examination Survey database. Progress in reducing the burden of lead toxicity is a public health success story that is incomplete with some identified factors posing larger, ongoing challenges.

Multiple Internal Hernias: A Complication of Laparoscopic Roux-en-Y Gastric Bypass.

Introduction: Internal hernias are the most common cause of small bowel obstruction following laparoscopic Roux-en-Y gastric bypass surgery (LRYGBP) with four distinct types. Herein, we report the clinical course of a patient with two independent hernias at the Petersen's space and a rarer subtype at the jejunojejunal window. A high index of suspicion for less common subtypes of internal hernias and the possibility of multiple, simultaneous internal hernias is critical. Case Description: We describe the case of a 52-year-old female with a history of LRYGBP who presented with abdominal pain and emesis due to an internal hernia at Peterson's defect, requiring subsequent laparoscopic repair. On postoperative day three, the patient presented again with recurrent abdominal pain and emesis. Repeat exploratory laparoscopy found a separate internal hernia involving the jejunojejunal window with the previously repaired Petersen's defect intact. Discussion: This case illustrates a unique scenario of a patient post-LRYGBP with multiple internal hernias at the Peterson's space and the less common jejunojejunal window, which was missed during the index surgery. Failure to identify simultaneous hernias may result in additional invasive intervention and further morbidity. Conclusion: Multiple less-common variants of internal hernias may present simultaneously following LRYGBP.

Engineering of HIV-1 neutralizing antibody CAP256V2LS for manufacturability and improved half life.

The broadly neutralizing antibody (bNAb) CAP256-VRC26.25 has exceptional potency against HIV-1 and has been considered for clinical use. During the characterization and production of this bNAb, we observed several unusual features. First, the antibody appeared to adhere to pipette tips, requiring tips to be changed during serial dilution to accurately measure potency. Second, during production scale-up, proteolytic cleavage was discovered to target an extended heavy chain loop, which was attributed to a protease in spent medium from 2-week culture. To enable large scale production, we altered the site of cleavage via a single amino acid change, K100mA. The resultant antibody retained potency and breadth while avoiding protease cleavage. We also added the half-life extending mutation LS, which improved the in vivo persistence in animal models, but did not impact neutralization activity; we observed the same preservation of neutralization for bNAbs VRC01, N6, and PGDM1400 with LS on a 208-virus panel. The final engineered antibody, CAP256V2LS, retained the extraordinary neutralization potency of the parental antibody, had a favorable pharmacokinetic profile in animal models, and was negative in in vitro assessment of autoreactivity. CAP256V2LS has the requisite potency, developability and suitability for scale-up, allowing its advancement as a clinical candidate.

In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs.

The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

Inhibition of experimental metastasis and extracellular matrix degradation by butanol extracts from B16-F1 murine melanoma.

We previously demonstrated that noncytolytic butanol extraction of B16 melanoma cells can increase the number of experimental lung metastases, and that brief incubation of the extracted cells with the extracted moieties reduces metastatic phenotype. This study examined the possibility that the extracted components are endogenous inhibitors of tumor cell surface-associated, degradative enzymes. The activity was found to be tumor associated, since only tumor extracts could reduce the number of experimental lung metastases of a variety of solid tumors. The activity in crude butanol extracts of B16-F1 that modulated the metastatic phenotype of extracted B16-F10 was partially purified by preparative isoelectric focusing and high-performance gel permeation chromatography. Incubation of extracted B16-F10 cells with low (Mr 2,000-10,000) molecular weight materials focusing in the pH 5.6 to 5.8 region of the preparative isoelectric focusing gradient significantly reduced the number of experimental lung foci. Ampholines alone had no effect. Evidence that the extracted moiety might be an endogenous enzyme inhibitor was obtained with the use of the subendothelial matrix degradation assay, wherein B16-F10 cells digest 35S-labeled heparan sulfate proteoglycan. The same materials that reduced the metastatic potential of butanol-extracted B16-F10 cells also inhibited extracellular matrix degradation by 30 to 85%, as well as the activity of partially purified heparanase (endo-beta-glucuronidase). The metalloproteinase inhibitor 1,10-phenanthroline and the heparanase inhibitor heparin partially (30 to 50%) blocked extracellular matrix degradation. Conversely, inhibitors of serine, thiol, acid, and other proteases had little or no effect on extracellular matrix degradation. These data provide evidence that an endogenous, heat-stable inhibitor of cell surface degradative enzymes such as heparanase may play a role in hematogenous metastasis, and support the hypothesis that butanol extraction activates some of these surface enzymes by removing the endogenous inhibitors.

Hepatic gene therapy: persistent expression of human alpha 1-antitrypsin in mice after direct gene delivery in vivo.

The liver represents an excellent target organ for gene therapy. The current strategy for hepatic gene therapy involves the isolation of primary hepatocytes from a resected liver lobe, transduction of therapeutic genes in vitro followed by autologous hepatocellular transplantation. This ex vivo approach is a rather complex procedure in its entirety; thus, a simple method for direct gene delivery into hepatocytes in vivo has been developed. The procedure involves partial hepatectomy followed by the portal vein infusion of recombinant retroviral vectors. Histological analysis of hepatocytes after in vivo delivery of a recombinant retrovirus bearing the E. coli beta-galactosidase gene showed that 1-2% of the parenchymal cells were transduced. Direct hepatic transfer of human alpha 1-antitrypsin cDNA under the transcriptional direction of the albumin promoter-enhancer led to constitutive expression of the human protein in the sera of recipients at concentrations of 30-1,400 ng/ml for at least 6 months. The experimental animals showed no signs of illness and histologic analysis of the liver revealed no evidence of pathologic abnormalities. The results suggest that the in vivo approach is an attractive alternative for hepatic gene therapy.

Effect of estrogen-promoted bacterial infections of the rat uterus on bioassay of mammalian cell growth factor activities in uterine luminal fluid.

Exogenous estradiol treatment of intact or ovariectomized rats causes accumulation of significant volumes of fluid in the uterine horns. In this report, evidence is presented showing the presence of mammalian cell growth factor(s) in uterine luminal fluid (ULF), along with other data showing that the exogenous estradiol treatment needed to cause significant accumulation of the fluid also facilitates the movement of vaginal origin bacteria into the uterine horns. It is shown that microorganisms infect the uteri of 80% or more of rats administered exogenous estradiol, and that the microorganisms are most probably of vaginal origin; procedures such as ligation of the uterine body above the cervix or antibiotic treatment did not suppress the infections. Administration of different doses of exogenous estrogen by either implantation of a single 25-mg estradiol/cholesterol pellet which causes a 20- to 50-fold elevation of estradiol levels above physiological plasma concentrations, or instead, by a Silastic tube delivery method that elevates levels only 2- to 3-fold above the normal range, resulted in equal frequency of uterine infections and in the appearance of infection at the same time after starting treatment. A number of bacterial species are present in the contaminated ULF, and these are the origins of intracellular products which are potent inhibitors of mammalian cell growth; the presence of these bacterial origin inhibitors interferes with the bioassay of the ULF growth factor activity, and hence, impedes the characterization of the growth factor(s) present in luminal fluid. Characterization of the origins of the growth-inhibiting activities showed that Pseudomonas aeruginosa and Proteus mirabilis are the predominant species present in infected uteri and that both produce exotoxin activities which inhibit growth of mammalian cells in culture; Pseudomonas appears to be the greater producer of cytotoxic activity. Evidence is presented that suggests that the well-known Exotoxin A produced by Pseudomonas may be responsible, in part, for the toxic effects of this organism. Other, as yet unidentified, cell growth inhibitors also may be produced by the bacteria found in ULF. Surgical separation of the uterine body from the cervix allows preparation of ULF which contains no bacteria and substantially reduced levels of growth inhibitors to mammalian cell lines.

Properties of a growth factor activity present in crude extracts of rat uterus.

We have shown previously (D. A. Sirbasku, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:3786-3790) that an estrogen-inducible growth factor activity for rat mammary and rat pituitary tumor cells can be identified in extracts of rat uteri, although at the time of that report only a limited biochemical characterization of the activity was presented. In this report, we have evaluated the growth factor activity for lipid, steroid hormone or protein-like properties. Uterine growth factor activity was assayed by measure of the increased cell number of the MTW9/PL rat mammary tumor cell line established by this laboratory and described previously (D. A. Sirbasku, 1978, Cancer Res. 38:1154-1165). Studies showed the following characteristics of growth factor activity: destroyed by trypsin treatment; labile when heated at 80 degrees C; partially denatured by 6 M guanidine or 8 M urea treatment or 50% aqueous solutions of organic solvents; inactivated by extremes of pH or overnight treatment with mild acid; not dialyzable at neutral pH; of apparent molecular weight of 70,000 daltons by G-100 Sephadex chromatography; possessing an isoelectric point of 4.8 to 5.2; not chloroform/methanol extractable; and not in any way identified as either a lipid or a steroid hormone. The data available suggest that the uterine growth factor activity is a protein or polypeptide of apparent high molecular weight, and that the activity does not directly correspond to other known growth factors.

Therapeutic serum concentrations of human alpha-1-antitrypsin after adenoviral-mediated gene transfer into mouse hepatocytes.

Alpha-1-antitrypsin is a relatively common genetic deficiency that results in early emphysema. The liver as the natural source of most alpha-1-antitrypsin synthesis was the target organ selected for gene replacement therapy studies. Previous work used recombinant retroviral vectors that encode the human alpha-1-antitrypsin cDNA for ex vivo and direct in vivo transduction of hepatocytes in dogs and rodents. This approach led to low levels of human protein in the serum of recipients. In this study, recombinant adenoviral vectors that express the human alpha-1-antitrypsin cDNA under the transcriptional control of the phosphoglycerate kinase (PGK) or RSV-LTR promoters have been constructed and used for the direct transduction of mouse hepatocytes in vivo. The animals transduced with the recombinant adenoviral vectors had therapeutic serum levels of human alpha-1-antitrypsin of up to 700 micrograms/mL. Thus, adenovirus-mediated gene transfer of the hAAT cDNA into the liver was able to produce therapeutic serum concentrations of protein.

Identification of estrogen-inducible growth factors (estromedins) for rat and human mammary tumor cells in culture.

The role of polypeptide growth factors (estromedins) as mediators of estrogen-responsive mammary tumor growth is studied in this report. Three possible new mechanisms were investigated that include endocrine, autocrine, and paracrine related growth factors. The first hypothesis being tested is whether estrogens interact with target tissues and cause the biosynthesis and secretion of polypeptide growth factors, which then act as mitogens for normal and neoplastic mammary tissues. Data presented suggest that this mechanism involves estrogen interaction with uterus, kidney, and pituitary gland causing production of growth factors, which then enter the general circulation and promote growth of distant target tissues. This is an endocrine type mechanism. Another type of estromedin control (autocrine control) may be exerted in an autostimulatory way in which the target tissue produces the polypeptide factors for its own growth in response to estrogen stimulation. A variation of the autocrine mechanism may be a paracrine mechanism in which some cells of an estrogen-responsive normal or neoplastic tissue produce growth factors that act on adjacent or neighboring cells. From the data available, all three possible types of growth factors could be functioning synergistically to yield the final result of continuous estrogen responsive tumor growth in vivo.

Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation.

One approach to gene therapy for hepatic diseases is to remove hepatocytes from an affected individual, genetically alter them in vitro, and reimplant them into a receptive locus. Although returning hepatocytes to the liver itself would be advantageous, the feasibility of this approach has never been evaluated due to the inability to distinguish donor from host hepatocytes. To unambiguously identify transplanted hepatocytes after transplantation, and to better quantitate their number and degree of liver function, two transgenic mouse lines were generated in a C57BL/6 background. The first expresses the Escherichia coli beta-galactosidase gene from the relatively liver-specific human alpha 1-antitrypsin (hAAT) promoter and allows transgenic hepatocytes to be readily identified after 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining; the second produces the hAAT protein under control of the same promoter, which enables hepatocyte survival and maintenance of liver function to be quantitated by measuring the serum levels of hAAT. Hepatocytes isolated from transgenic donors were transplanted into nontransgenic C57BL/6 recipients by intrasplenic injection. Surprisingly, a large fraction of these cells were identified within the liver parenchyma but not the spleen at 2 months after transplantation. The high levels of serum hAAT detected in transplant recipients were stable for greater than 6 months, suggesting that established cells will survive indefinitely. These results have important implications for liver organogenesis and hepatic gene therapy.

In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs.

Hemophilia B is a bleeding disorder caused by mutations in the factor IX gene. The disorder is X-linked recessive with a prevalence of about 1 in 30,000 Caucasian males. Factor IX is naturally synthesized in the liver and secreted into blood. Here we report the construction of recombinant adenoviral vectors containing the canine factor IX cDNA that are capable of transducing hepatocytes in mice at high efficiencies in vivo without partial hepatectomy. The recombinant viral vector was used to treat hemophilia B dogs by direct vector infusion into the portal vasculature of deficient animals. Plasma factor IX concentrations in the treated hemophilia B dogs increased from 0 to 300% of the level present in normal dogs, resulting in complete amelioration of the disease as demonstrated by normal blood coagulation and hemostatic measurements. Although plasma factor IX concentration started to decline after a few days, therapeutic levels of factor IX persisted for 1-2 months in the treated animals. The results validate the principle of in vivo hepatic gene delivery to reconstitute the genetic deficiency in a large animal model and suggest that gene therapy is achievable when long-acting vectors are developed.

A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells.

To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.

Expression of human alpha 1-antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes.

The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. We have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here we demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. At least 1 x 10(9) hepatocytes or 5% of the liver mass can be transplanted by the portal vasculature. In two animals we have transplanted hepatocytes transduced with a retroviral vector containing the human alpha 1-antitrypsin cDNA under transcriptional control of the cytomegalovirus promoter. Both animals had significant human alpha 1-antitrypsin in the serum for 1 month. Although the serum levels of human alpha 1-antitrypsin eventually fell due to inactivation of the cytomegalovirus promoter, PCR analysis demonstrated that a significant fraction of transduced hepatocytes migrated to the liver and continued to survive in vivo. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstitution with the use of promoters of cellular genes that are active in the normal liver.