High-yield actinorhodin production in fed-batch culture by a Streptomyces lividans strain overexpressing the pathway-specific activator gene actll-ORF4.

Streptomyces lividans 1,326 usually does not produce the red/blue colored polyketide actinorhodin in liquid culture even though it carries the entire actinorhodin biosynthesis gene cluster. The bacterium can be forced to produce this secondary metabolite by introducing actII-ORF4, the actinorhodin pathway-specific activator gene from Streptomyces coelicolor, on a multicopy plasmid. The production of actinorhodin by such a strain has been optimized by medium and process manipulations in fed-batch cultures. With high-yield cultivation conditions, 5 g actinorhodin/l are produced during 7 days of cultivation; or approximately 0.1 g actinorhodin/g dry weight (DW)/day in the production phase. The yield in this phase is 0.15 Cmol actinorhodin/Cmol glucose, which is in the range of 25% to 40% of the maximum theoretical yield. This high-level production mineral medium is phosphate limited. In contrast, nitrogen limitation resulted in low-level production of actinorhodin and high production of a-ketoglutaric acid. Ammonium as nitrogen source was superior to nitrate supporting an almost three times higher actinorhodin yield as well as a two times higher specific production rate. The wild-type strain lacking the multicopy plasmid did not produce actinorhodin when cultivated under any of these conditions. This work examines the actinorhodin-producing potential of the strain, as well as the necessity to improve the culture conditions to fully utilize this potential. The overexpression of biosynthetic pathway-specific activator genes seems to be a rational first step in the design of secondary metabolite overproducing strains prior to alteration of primary metabolic pathways for redirection of metabolic fluxes.

pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis.

AIMS: To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. METHODS AND RESULTS: The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6.5 to 9.25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6.5, growth at pH 9.25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6.5. CONCLUSIONS: A change in the operating pH from 6.5 to 9.25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release.

Measurement of continuous ambulatory peritoneal dialysis prescription adherence using a novel approach.

OBJECTIVE: The purpose of the study was to test a novel approach to monitoring the adherence of continuous ambulatory peritoneal dialysis (CAPD) patients to their dialysis prescription. DESIGN: A descriptive observational study was done in which exchange behaviors were monitored over a 2-week period of time. SETTING: Patients were recruited from an outpatient dialysis center. PARTICIPANTS: A convenience sample of patients undergoing CAPD at Piedmont Dialysis Center in Winston-Salem, North Carolina was recruited for the study. Of 31 CAPD patients, 20 (64.5%) agreed to participate. MEASURES: Adherence of CAPD patients to their dialysis prescription was monitored using daily logs and an electronic monitoring device (the Medication Event Monitoring System, or MEMS; APREX, Menlo Park, California, U.S.A.). Patients recorded in their logs their exchange activities during the 2-week observation period. Concurrently, patients were instructed to deposit the pull tab from their dialysate bag into a MEMS bottle immediately after performing each exchange. The MEMS bottle was closed with a cap containing a computer chip that recorded the date and time each time the bottle was opened. RESULTS: One individual's MEMS device malfunctioned and thus the data presented in this report are based upon the remaining 19 patients. A significant discrepancy was found between log data and MEMS data, with MEMS data indicating a greater number and percentage of missed exchanges. MEMS data indicated that some patients concentrated their exchange activities during the day, with shortened dwell times between exchanges. Three indices were developed for this study: a measure of the average time spent in noncompliance, and indices of consistency in the timing of exchanges within and between days. Patients who were defined as consistent had lower scores on the noncompliance index compared to patients defined as inconsistent (p = 0.015). CONCLUSIONS: This study describes a methodology that may be useful in assessing adherence to the peritoneal dialysis regimen. Of particular significance is the ability to assess the timing of exchanges over the course of a day. Clinical implications are limited due to issues of data reliability and validity, the short-term nature of the study, the small sample, and the fact that clinical outcomes were not considered in this methodology study. Additional research is needed to further develop this data-collection approach.

The Kidney Outcomes Prediction and Evaluation (KOPE) study: a prospective cohort investigation of patients undergoing hemodialysis. Study design and baseline characteristics.

PURPOSE: The purpose of the Kidney Outcomes Prediction and Evaluation (KOPE) study, was to more fully characterize the end-stage renal disease (ESRD) population with respect to social, psychological, and clinical characteristics, and to prospectively study the biomedical, social, and psychological factors that influence a range of ESRD outcomes in a large observational study of black and white patients on hemodialysis. This paper focuses on the KOPE study design as well as characteristics of patients at baseline. METHODS: KOPE was a prospective cohort investigation of patients treated at four dialysis centers in Forsyth County, North Carolina. Participants were interviewed at the dialysis centers, semi-annually over a 3 1/2 year period. Prevalent cases who were being treated with hemodialysis at the initiation of the study were enrolled into KOPE. Incident cases were subsequently enrolled as they presented to the participating units for hemodialysis. A total of 304 prevalent and 162 incident cases were enrolled into the study. The baseline health and sociodemographic characteristics of KOPE participants reported in this paper were obtained from medical records and Southeast Kidney Council data. Laboratory values taken within a 30-day interval around the baseline interview are also reported. RESULTS: KOPE participants differ from national statistics on race, age, and gender. Differences between KOPE participants and patients living in the region, but who did not participate in the study, can be explained by our recruitment criteria. CONCLUSIONS: KOPE will enable the characterization of the ESRD population, identification of factors related to poor outcomes, and identification of opportunities for interventions to prevent death and morbidity.

Predictors of crop diversification: a survey of tobacco farmers in North Carolina (USA).

OBJECTIVE: To assess the attitudes and behaviours of North Carolina tobacco farmers around crop diversification. DESIGN: Cross-sectional telephone survey. PARTICIPANTS: Active tobacco farmers in 14 North Carolina counties (n = 1236), interviewed between January and April 1997 (91% response rate). OUTCOME MEASURES: Interest in, experience with, and perceived barriers to diversification. RESULTS: Most farmers (95%) grew/raised a commodity other than tobacco (mean = 2.8). A total of 60% of farmers expressed interest in trying other on-farm activities to supplement their tobacco and 60% reported taking action in the past year around supplementation. Younger age and college education were positively associated with interest. College education, off-farm income, and larger farm size were associated with the number of actions taken. For perceived external barriers to diversification, use of tobacco, percent income from tobacco, lack of college education, and younger age were most strongly associated with the number of barriers. For internal barriers (personal factors), percent income from tobacco, use of tobacco, and lack of college education were most strongly associated with the number of barriers. CONCLUSIONS: Most farmers were involved in diverse operations and expressed interest in continuing to diversify, although the breadth of diversification was narrow. Farmers noted many barriers to diversifying. If conventional production and marketing techniques are employed for non-tobacco alternatives, these alternatives may not provide the sustainable profitability that tobacco has afforded. Competition from foreign tobacco growers is the primary threat to the future of American growers and tobacco dependent communities.

The early use of alcohol and tobacco: its relation to children's competence and parents' behavior.

OBJECTIVES: Use of tobacco and alcohol during childhood predicts heavy use of these substances and use of illicit drugs during adolescence. This study aims to identify developmental correlates of tobacco and alcohol use among elementary-school children. METHODS: Cross-sectional surveys were used to measure tobacco and alcohol use, multiple indicators of child competence, parenting behaviors, and parental modeling of tobacco and alcohol use in a sample of 1470 third- and fifth-grade children. Both self-report and teacher-rated assessments were obtained, which allowed collateral testing of study hypotheses. RESULTS: Children's tobacco and alcohol use was strongly related to low scores on several measures of child competence, both self-reported and teacher rated. Children's tobacco and alcohol use was also associated with less effective parenting behaviors and with parental use of tobacco and alcohol. CONCLUSIONS: Children's early experience with tobacco and alcohol is associated with weak competence development and exposure to socialization factors that promote risk taking. Interventions to prevent early use of tobacco and alcohol are needed.

Tobacco promotion and susceptibility to tobacco use among adolescents aged 12 through 17 years in a nationally representative sample.

OBJECTIVES: The purpose of this study was to examine whether youth participation in tobacco promotion campaigns is associated with susceptibility to tobacco use. METHODS: Data were collected from telephone interviews of a national random sample of 1047 adolescents 12 to 17 years of age. RESULTS: A proportional odds model was used to estimate the effects of age, gender, presence of a tobacco user in the household, awareness of tobacco promotions, knowledge of a young adult or adolescent friend owning a promotional item, participation in tobacco promotions, and receipt of free tobacco samples or direct mail from tobacco companies on susceptibility to tobacco use. All of the covariates, except for receiving direct mailings and knowing a young adult friend who owned a promotional item, were significantly associated with susceptibility. CONCLUSIONS: There is a strong association between an awareness of and involvement with tobacco promotions and being susceptible to tobacco use or a user of tobacco products.

Tobacco farmers and diversification: opportunities and barriers.

OBJECTIVE: To assess the knowledge, attitudes, and behaviours of tobacco growers and allotment owners in the southeastern United States. DESIGN: Cross-sectional telephone survey. PARTICIPANTS: Tobacco growers (n = 529) and tobacco allotment owners (n = 417) were interviewed by telephone in March 1995. SETTING: Tobacco growing states in the southeastern US. MAIN OUTCOME MEASURES: Attitudes of tobacco growers and tobacco allotment owners towards, and experience with, diversification; and attitudes towards an increase in the federal excise tax on tobacco. RESULTS: Half of the respondents had done something to learn about on-farm alternatives to tobacco, had an interest in trying other on-farm ventures to supplement tobacco income, and found alternatives that were profitable. There was a strong, negative linear trend between age and being interested in or trying alternative enterprises. Structural and economic impediments to diversification were noted by respondents (especially younger respondents), but 73% supported an increase in the federal excise tax on tobacco if the money was used to help farmers overcome these barriers. CONCLUSIONS: These data suggest that farmers and health professionals have reason to establish dialogue around diversification and using excise tax increases to fund diversification and to promote health. Tobacco companies have been successful in mobilising farmers against tax increases, but efforts must be made to show farmers that tax increases can be beneficial both to their diversification efforts and to public health. The outcome of this dialogue may well affect the economic infrastructure of thousands of rural communities, the livelihood of tens of thousands of tobacco farmers and their families, and the health of millions of tobacco users.

Effect of integration of injury control information into a high school physics course.

STUDY OBJECTIVE: To investigate the effects on knowledge, attitudes, and self-reported behaviors of a 1-week course of injury control and crash safety information integrated within a high school physics curriculum. METHODS: Students in an intervention high school (n=129) were compared with students in a control high school (n=74) enrolled in a comparable physics curriculum. A standardized survey was administered before instruction (time T1), and at 2 weeks (T2) and 6 months (T3) after instruction was completed. The behaviors measured were self-reported use of seat belts, speeding, drinking and driving, and intention to use seat belts in the future. RESULTS: At T2, students in the intervention group reported attitudes that were less favorable toward risk-taking in regard to speeding and seat belt use than those of the control group. At T3, there was still a difference in attitudes toward speeding but not toward seat belt use. The intervention significantly altered the knowledge level of the course participants, and these changes persisted to T3. The strongest and most persistent change was that students in the intervention group reported increasing their use of seat belts when riding as a passenger. (Seat belt use as a driver was high for both groups.) The intervention group showed a significant increase in their 1-year intentions to use seat belts both as a driver and as a passenger. CONCLUSION: This study demonstrated that driver safety education can be successfully integrated into a mainstream high school science curriculum. Future studies measuring the effects of this curriculum on observed behaviors are needed.

The effective diffusion coefficient and the distribution constant for small molecules in calcium-alginate gel beads.

The effective diffusion coefficient, D(e), and the distribution constant, K(i), for selected mono- and disaccharides and organic acids were determined in homogeneous calcium-alginate gel with and without entrapped bacteria. Results were obtained from transient concentration changes in well-stirred solutions of limited volume, in which the gel beads were suspended. The effective diffusioncoefficients and the distribution constants were estimated by fitting mathematical model predictions to the experimental data using a nonlinear model fitting program (MODFIT). Both single solute diffusion and multiple solute diffusion were performed. A small positive effect was obtained onthe values of D(e) for the system of multiple solute diffusion; however, the values of K(i) were not significantly influenced. For the nine solutes tested, D(e) for 2% Ca-alginate gel beads was found to be approximately 85% of the diffusivity measured in water. The effects on D(e) and K(i), for lactose and lactic acid were determined for variations of alginate concentration, pH, temperature, and biomass content in the beads. D(e) decreased linearly for both lactose and lactic acid with increasing cell concentration in the Ca-alginate gel. K(i), was constant for both lactose and lactic acid with increasing cell concentration. D(e) was significantly lower at pH 4.5 than at pH 5.5 and 6.5 for both lactose and lactic acid. Furthermore, D(e) seemed to decrease with increased alginate concentration in the range of 1% to 4%. The diffusion rate increased with increasing temperature, and the activation energy for the diffusion process for both lactose and lactic acid was constant in the temperature range tested. (c) 1995 John Wiley & Sons Inc.

Determination of effective diffusion coefficients and distribution constants in polysaccharide gels with non-steady-state measurements.

A non-steady-state method has been used for determining the effective diffusion coefficient, D(e), and a distribution constant, K(i), of small molecules in alginate gel beads. A mathematical model based on Pick's law and includingexternal film diffusion resistance describe the diffusion process. Criticalexperimental parameters for the estimation of D(e) and K(i), for both one- and two-parameter methods were the initial solute concentration in the bulk liquid, the void fraction inthe reactor, and the experimental starting point. In our analysis, the two-parameter method is preferable. Incorporation of an estimate of the film resistance into the overall model increased the estimated values of D(e) significantly and improved the stability of the term over a range of reactor agitation rates. (c) 1995 John Wiley & Sons Inc.

Properties of a recombinant human uracil-DNA glycosylase from the UNG gene and evidence that UNG encodes the major uracil-DNA glycosylase.

We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.

Transport of osmoprotective compounds in hybridoma cells exposed to hyperosmotic stress.

Addition of osmoprotective compounds has a positive effect on growth and monoclonal antibody production in hyperosmotic hybridoma cell cultures. In order to better understand the processes involved in the osmoprotective response, uptake of the osmoprotective compounds glycine betaine, proline, sarcosine and glycine in mouse hybridoma cell line 6H11 during exposure to hyperosmotic stress was studied. Hyperosmotic stress (510 mOsmol/kg) was introduced through the addition of NaCl (100 mM) to the growth medium, and amino acid transport activity was measured immediately after transfer of the cells to the hyperosmotic medium. The osmoprotective capability of the four osmoprotectants tested was negatively affected if methylaminosobutyric acid (MeAiB), a specific substrate for amino acid transport system A, was simultaneously included in the hyperosmotic medium in equimolar amounts with one of the osmoprotective compounds. This was due to accumulation of MeAiB in the stressed cells, giving a significant reduction in the concentration of the osmoprotective compound inside the cells. Furthermore, addition of excess meAiB gave approx. 905 reduction in the initial rate of uptake of glycine betaine, while 40-50% reduction in the initial rate of uptake of proline, glycine and sarcosine. Similarly, addition of proline, glycine or sarcosine also gave a significant reduction in the initial rate of glycine betaine uptake. These results suggest that the four osmoprotective compounds share, at least in part, a common, MeAiB inhibitable carrier for transport into osmotically stressed hybridoma cells. This carrier is probably equal to amino acid transport system A.

Hyperosmotic hbridoma cell cultures: Increased monoclonal antibody production with addition of glycine betaine.

When mouse hybridoma cells were grown in culture media which were made hyperosmotic through the addition of NaCl or sucrose, the specific rate of antibody production increased with medium osmolality, reaching approx. 1.9 times the level obtained at physiological osmolality. However, due to a simultaneous reduction of the maximal cell density in the hyperosmotic media, the effect of the increased production rate did not give significant increases in the maximum antibody titer obtained in the cultures. When the osmoprotective compound, glycine betaine, was included in the NaCl- or sucrose-stressed cultures, the specific antibody production rate wasincreased up to 2.6-fold and maximum antibody titer up to twofold over that obtained in the control culture (physiological osmolality). A similar pattern of response was observed when other osmoprotective compounds (sarcosine, proline, glycine) were added to NaCl-stressed hybridoma cell cultures. For the present experiments, the results suggest that medium osmolality, rather than growth rate, will determine the specific antibody production rate by hybridoma cell line 6H11 growing in hyperosmotic culture media. (c) 1994 John Wiley & Sons, Inc.

Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress.

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.

A self-administered questionnaire for the assessment of severity of symptoms and functional status in carpal tunnel syndrome.

We developed a self-administered questionnaire for the assessment of severity of symptoms and functional status in patients who have carpal tunnel syndrome. The reproducibility, internal consistency, validity, and responsiveness to clinical change of scales for the measurement of severity of symptoms and functional status were evaluated in a clinical study. The scales were highly reproducible (Pearson correlation coefficient, r = 0.91 and 0.93 for severity of symptoms and functional status, respectively) and internally consistent (Cronbach alpha, 0.89 and 0.91 for severity of symptoms and functional status, respectively). Both scales had positive, but modest or weak, correlations with two-point discrimination and Semmes-Weinstein monofilament testing (Spearman coefficient, r = 0.12 to 0.42). In thirty-eight patients who were operated on in 1990 and were evaluated a median of fourteen months postoperatively, the mean symptom-severity score improved from 3.4 points preoperatively to 1.9 points at the latest follow-up examination, while the mean functional-status score improved from 3 to 2 points (5 points is the worst score and 1 point is the best score for each scale). Similar improvement was noted in twenty-six patients who were evaluated before and three months after the operation. We concluded that the scales for the measurement of severity of symptoms and functional status are reproducible, internally consistent, and responsive to clinical change, and that they measure dimensions of outcomes not captured by traditional measurements of impairment of the median nerve. These scales should enhance standardization of measurement of outcomes in studies of treatment for carpal tunnel syndrome.

Safety of freeway median high occupancy vehicle lanes: a comparison of aggregate and disaggregate analyses.

This paper addresses safety issues associated with High Occupancy Vehicle (HOV) lanes constructed along freeway medians, without physical separation from adjacent traffic. Data associated with operation of such an HOV facility in Southern California are analyzed relative to the pattern of accidents on the facility and the potential role of congestion. Detailed analyses of accident characteristics point out that potentially false conclusions regarding the safety of HOV lanes can be drawn from simple analyses that are based on aggregate measures of accident frequencies and assumed traffic volumes.

Comparison of the Matthews Youth Test for Health and the Hunter-Wolf A-B Rating Scale: measures of type A behavior in children.

This study evaluated the extent to which two youth measures of Type A behavior--the Matthews Youth Test for Health (MYTH) and the Hunter-Wolf A-B Rating Scale--similarly assess the Type A construct. Data from 25 elementary teachers and from 300 of their students revealed that these scales are weakly correlated and that the concordance of their Type A-Type B classifications was only slightly above that expected by chance. Weak agreement was found even when teachers and students rated the same Type A behaviors, which suggests that variability in content was not the principal reason for the lack of agreement between these measures. The major implication of this study is that the MYTH and the Hunter-Wolf scale should not be considered interchangeable measures of Type A behavior. The study reveals that investigators' choice of a Type A measure may strongly affect the nature of their research findings and may inhibit the integration of their data with data from other investigators who employ different measures of the Type A construct. It is recommended that, if multiple measures cannot be used in a study, the investigator employ the MYTH.