Nature of fiber-coupled detector responsivity measurements at 0.1% using a primary standard.

We demonstrate the capability to measure the absolute power responsivity of optical fiber-coupled detectors at an expanded uncertainty of 0.1%, by direct comparison with a cryogenic primary standard. To facilitate synchronous power measurements, commercial all-fiber beam-splitters direct laser diode light simultaneously to the device under test and the primary standard. We investigate the use of single-mode, polarisation maintaining, and photonic crystal fibers to access the cryogenic standard, and report a reduction in the temperature dependent effective refractive index of these fibers of 0.1%, 0.15% and 0.3% respectively in going from room temperature to 5 K. We also evaluate the polarisation dependent loss of the beam-splitters, the stability of the beam-splitter ratio between the cryogenic detector and the device under test and the temporal and modal stability of the Fabry-Pérot laser diode sources. It is shown that the stability of the optical fiber beam-splitters limits the overall performance of the measurement system to an expanded uncertainty of 0.1%.

A conceptual framework for an oral cancer awareness campaign in Northern Germany - Challenges in campaign development and assessment.

Initial impetus for action: Oral cancer is still an underestimated disease in terms of incidence as well as mortality rates; it requires urgent prevention and early detection. At present, there is no best-practice systematic approach to raising awareness and informing the public about about this type of cancer in Germany. This article describes a framework that covers the significant stages of conceptual development and campaign design to promote oral cancer awareness in Germany. Solution: The challenges of the development, as well as evaluation of an oral cancer awareness campaign are shared in this article. Four key stages of the campaign are defined: (1) mass media, (2) target groups, (3) health care professionals, and (4) epidemiology. For each section, the following levels of assessment are proposed: (a) campaign development (formative assessment), (b) controlling and optimising campaign implementation (process assessment) and (c) measuring outcomes (summative assessment). Outcome: A process-oriented assessment concept for each of the four campaign sections was developed and merged to form a matrix, which includes each of the above sections regarding the prevention and early detection of oral cancer, as well as the three stages of campaign assessment. Future implications and learning points: The conceptual framework demonstrated that systematic planning and evaluation of different components helped to describe and evaluate an oral cancer campaign: For future campaigns, the use of a matrix covering different campaign targets as well as the entire campaign process, is recommended as a basis for campaign design and evaluation.

Particle and vapor emissions from vat polymerization desktop-scale 3-dimensional printers.

Little is known about emissions and exposure potential from vat polymerization additive manufacturing, a process that uses light-activated polymerization of a resin to build an object. Five vat polymerization printers (three stereolithography (SLA) and two digital light processing (DLP) were evaluated individually in a 12.85 m3 chamber. Aerosols (number, size) and total volatile organic compounds (TVOC) were measured using real-time monitors. Carbonyl vapors and particulate matter were collected for offline analysis using impingers and filters, respectively. During printing, particle emission yields (#/g printed) ranged from 1.3 ± 0.3 to 2.8 ± 2.6 x 108 (SLA printers) and from 3.3 ± 1.5 to 9.2 ± 3.0 x 108 (DLP printers). Yields for number of particles with sizes 5.6 to 560 nm (#/g printed) were 0.8 ± 0.1 to 2.1 ± 0.9 x 1010 and from 1.1 ± 0.3 to 4.0 ± 1.2 x 1010 for SLA and DLP printers, respectively. TVOC yield values (µg/g printed) ranged from 161 ± 47 to 322 ± 229 (SLA printers) and from 1281 ± 313 to 1931 ± 234 (DLP printers). Geometric mean mobility particle sizes were 41.1-45.1 nm for SLA printers and 15.3-28.8 nm for DLP printers. Mean particle and TVOC yields were statistically significantly higher and mean particle sizes were significantly smaller for DLP printers compared with SLA printers (p < 0.05). Energy dispersive X-ray analysis of individual particles qualitatively identified potential occupational carcinogens (chromium, nickel) as well as reactive metals implicated in generation of reactive oxygen species (iron, zinc). Lung deposition modeling indicates that about 15-37% of emitted particles would deposit in the pulmonary region (alveoli). Benzaldehyde (1.0-2.3 ppb) and acetone (0.7-18.0 ppb) were quantified in emissions from four of the printers and 4-oxopentanal (0.07 ppb) was detectable in the emissions from one printer. Vat polymerization printers emitted nanoscale particles that contained potential carcinogens, sensitizers, and reactive metals as well as carbonyl compound vapors. Differences in emissions between SLA and DLP printers indicate that the underlying technology is an important factor when considering exposure reduction strategies such as engineering controls.

The Role of Sulfur Dioxide in Stratospheric Aerosol Formation Evaluated Using In-Situ Measurements in the Tropical Lower Stratosphere.

Stratospheric aerosols (SAs) are a variable component of the Earth's albedo that may be intentionally enhanced in the future to offset greenhouse gases (geoengineering). The role of tropospheric-sourced sulfur dioxide (SO2) in maintaining background SAs has been debated for decades without in-situ measurements of SO2 at the tropical tropopause to inform this issue. Here we clarify the role of SO2 in maintaining SAs by using new in-situ SO2 measurements to evaluate climate models and satellite retrievals. We then use the observed tropical tropopause SO2 mixing ratios to estimate the global flux of SO2 across the tropical tropopause. These analyses show that the tropopause background SO2 is about 5 times smaller than reported by the average satellite observations that have been used recently to test atmospheric models. This shifts the view of SO2 as a dominant source of SAs to a near-negligible one, possibly revealing a significant gap in the SA budget.

Interaction of potassium carbonate and soybean oil supplementation on performance of early-lactation dairy cows fed a high-concentrate diet.

Potassium carbonate supplementation is known to improve milk fat synthesis and to modify milk mineral composition in dairy cows. The objective of the current experiment was to evaluate the effect of K2CO3 on production performance, biohydrogenation of fatty acids (FA), and mineral composition of milk in early-lactation dairy cows fed a high-concentrate diet with or without soybean oil (SBO), as a source of polyunsaturated FA. Twenty-eight ruminally fistulated Holstein cows were used in a randomized complete block design. The experiment lasted 33 d, including a 5-d pretreatment collection period used as a covariate. Experimental treatments were arranged as a 2 × 2 factorial with 0 or 1.5% K2CO3 and with 0 or 2% SBO, and balanced to contain 40% forage (57% corn silage + 43% grass silage) and 60% concentrate. Preplanned orthogonal contrasts were used to assess the effects of K2CO3, SBO, and their interaction. Feeding K2CO3 did not affect milk yield, but tended to increase 4% fat-corrected milk and fat yield when combined with SBO. However, adding SBO to diets increased milk yield. Dietary K2CO3 supplementation did not affect milk fat concentration of trans-10 18:1 or any other identified biohydrogenation intermediates. Soybean oil supplementation decreased milk fat concentration of C16 and de novo synthesized FA, and increased preformed FA. Among the other effects of SBO supplementation observed, concentrations of cis-9,trans-11 18:2 increased, as well as most of the cis and trans isomers of 18:1 and 18:0. Milk urea N decreased in cows fed K2CO3 as compared with unsupplemented diets. A positive relation was established between milk Cl concentration and milk yield, suggesting that the equilibrium of this ion is linked to the efficiency of lactogenesis. The effect of K2CO3 on this mineral equilibrium in the mammary gland remains to be established. Overall, results have shown that potential effect of K2CO3 on milk fat synthesis is dependent on the levels of dietary polyunsaturated FA.

Effect of lipid supplementation on milk odd- and branched-chain fatty acids in dairy cows.

Eight ruminally fistulated, multiparous Holstein cows were arranged in a double 4×4 Latin square with 14-d periods to investigate the effects of lipid supplementation on performance, rumen parameters, the milk odd- and branched-chain fatty acid (OBCFA) profile, and the relationships between milk OBCFA and rumen parameters. Lipid supplementation is known to inhibit microbial growth in the rumen, decrease de novo microbial fatty acid synthesis, and increase the uptake of circulating fatty acids by the mammary gland; treatments were selected to isolate these effects on the milk OBCFA profile. The 4 treatments were (1) a lipid-free emulsion medium infused in the rumen (CTL), (2) soybean oil as a source of polyunsaturated fatty acids infused in the rumen (RSO), (3) saturated fatty acids (38% 16:0, 40% 18:0) infused in the rumen (RSF), and (4) saturated fatty acids infused in the abomasum (ASF). Fat supplements were provided continuously as emulsions at a rate of 450g/d. Preplanned contrasts compared CTL to RSO, RSO to RSF, and RSF to ASF. Infusing RSO slightly decreased ruminal pH, but did not affect volatile fatty acids profile and milk fat concentration as compared with CTL. The yields of energy-corrected milk, fat, and protein were greater with RSF compared with RSO. The concentration of odd-chain fatty acids was decreased by RSO, whereas even-chain iso fatty acids were not affected. Milk fat concentration of 17:0 + cis-9 17:1 was higher for RSF than for RSO, due to the saturated fatty acids supplement containing 2% 17:0 + cis-9 17:1. Limited differences were observed in the milk OBCFA profile between RSF and ASF. A multiple regression analysis yielded the following equation for predicting rumen pH based on milk fatty acids: pH=6.24 - (0.56×4:0) + (1.67 × iso 14:0) + (4.22 × iso 15:0) + (9.41×22:0). Rumen propionate concentration was negatively correlated with milk fat concentration of iso 14:0 and positively correlated with milk 15:0, whereas the acetate-to-propionate ratio gave the opposite correlations with milk iso 14:0 and 15:0. Milk fat concentration of 17:0 + cis-9 17:1 was not related to rumen propionate or to acetate-to-propionate ratio, due to the presence of 17:0 and cis-9 17:1 in the saturated fatty acids supplement. The results suggest that although lipid supplementation can affect the profile of milk OBCFA, the promise remains of using these milk fatty acids to evaluate rumen function.

Comb-calibrated laser ranging for three-dimensional surface profiling with micrometer-level precision at a distance.

Non-contact surface mapping at a distance is interesting in diverse applications including industrial metrology, manufacturing, forensics, and artifact documentation and preservation. Frequency modulated continuous wave (FMCW) laser detection and ranging (LADAR) is a promising approach since it offers shot-noise limited precision/accuracy, high resolution and high sensitivity. We demonstrate a scanning imaging system based on a frequency-comb calibrated FMCW LADAR and real-time digital signal processing. This system can obtain three-dimensional images of a diffusely scattering surface at stand-off distances up to 10.5 m with sub-micrometer accuracy and with a precision below 10 µm, limited by fundamental speckle noise. Because of its shot-noise limited sensitivity, this comb-calibrated FMCW LADAR has a large dynamic range, which enables precise mapping of scenes with vastly differing reflectivities such as metal, dirt or vegetation. The current system is implemented with fiber-optic components, but the basic system architecture is compatible with future optically integrated, on-chip systems.

Development of a dynamic model for ventral hernia mesh repair.

INTRODUCTION: The adequate way of mesh fixation in laparoscopic ventral hernia repair is still subject to debate. So far, simulation has only been carried out in a static way, thereby omitting dynamic effects of coughing or vomiting. We developed a dynamic model of the anterior abdominal wall. MATERIALS AND METHODS: An aluminium cylinder was equipped with a pressure controlled, fluid-filled plastic bag, simulating the abdominal viscera. A computer-controlled system allowed the control of influx and efflux, thus creating pressure peaks of up to 200 mmHg to simulate coughing and 290 mmHg to simulate vomiting. We tested fixation with tacks (Absorbatack, Covidien Deutschland, Neustadt a. D., Germany). The model was controlled for the friction coefficient of the tissue against the mesh and the physiologic elasticity of the abdominal wall surrogate. RESULTS: The model was able to create pressure peaks equivalent to physiologic coughs or vomiting. Physiologic elasticity was thereby maintained. We could show that the friction coefficient is crucial to achieve a physiologic situation. The meshes showed a tendency to dislocate with an increasing number of coughs (Fig. 4). Nevertheless, when applied in a plain manner, the meshes withstood more cough cycles than when applied with a bulge as in laparoscopic surgery. CONCLUSIONS: The dynamic movement of the abdominal wall, the friction between tissue and mesh and the way of mesh application are crucial factors that have to be controlled for in simulation of ventral abdominal hernia closure. We could demonstrate that patient specific factors such as the frequency of coughing as well as the application technique influence the long term stability of the mesh.

[Prevention of skin cancer: considerations on strategic communication]. / Prävention von Hautkrebs : Kommunikationsstrategische Überlegungen.

In recent decades the numbers of cases of skin cancer have been increasing worldwide in light skinned populations. In Germany skin cancer is the most common form of cancer. To reduce the burden of skin cancer protection from ultraviolet radiation (primary prevention) and early detection (secondary prevention) of the disease play a decisive role. In this context information to the population about preventive behavior and the support of informed decision-making in skin cancer screening are important aspects in communication. This paper gives an overview about communicational aspects in the promotion of skin cancer prevention. In the development of communicational interventions it is important to identify the relevant target groups. Relevant key opinion leaders have to be included in the information process. Additionally, interventions should be based on a theoretical framework and be designed for the respective target group. Furthermore, different forms of communication and communication tools are provided for the realization of an information intervention. To appraise the intervention elements of summative and formal evaluation are available. The current results provide important findings about different effects of communicational aspects on knowledge and behavior of the population; however, due to the complexity of information interventions a particular effect cannot be explained by a single communicational element.

The rise in acceptance of mental health professionals: help-seeking recommendations of the German public 1990-2020.

AIMS: We will first examine whether seeking help for depression and schizophrenia from mental health professionals is nowadays more accepted among the German public than it used to be 30 years ago. Next, we will explore whether changes in help-seeking preferences between 1990 and 2020 are specific to mental health professions or are part of changes in attitudes to professional help-seeking in general. Finally, we will study whether a temporal relationship does exist between the advent of awareness-raising and anti-stigma campaigns after the turn of the millennium and changes in the acceptance of mental health care. METHODS: In 1990 (n = 2044), 2001 (n = 4005), 2011 (n = 1984) and 2020 (n = 2449) methodologically identical population-based surveys were conducted in Germany. After presentation of an unlabelled case vignette depicting someone with either schizophrenia or depression, we asked about help-seeking recommendations for the person described. RESULTS: The German public's readiness to recommend seeking help from mental health professionals has markedly grown over the past 30 years. In contrast, in the eyes of the public, turning to a general practitioner has become only slightly more, consulting a priest even less advisable than it used to be three decades ago. Seeing a naturopath is seen with markedly less disapproval today compared to 1990, but explicit recommendation of this helping source has not increased correspondingly in. The most pronounced increase in the German public's propensity to recommend seeking help from mental health professionals occurred already in the 1990s, i.e. before efforts to heighten public awareness had started. CONCLUSIONS: Today, the German public is more in favour of mental health professionals than it used to be three decades ago. This seems to be a specific trend, and not to reflecting an increasing propensity towards professional help-seeking in general. Our findings counter the narrative that mental health communication efforts and initiatives have created more favourable attitudes towards mental health care among the public, since the observed changes in attitudes have preceded any campaigns. Instead, we tend to interpret the rise of the popularity of mental health professionals as a reflection of general cultural changes that have taken place over the past decades in Germany, as in other western countries.

Do values and political attitudes affect help-seeking? Exploring reported help-seeking for mental health problems in a general population sample using a milieu framework.

AIMS: Help-seeking for mental health problems is facilitated and hindered by several factors at the individual, interpersonal and community level. The most frequently researched factors contributing to differences in help-seeking behaviour are based on classical socio-demographic variables, such as age, gender and education, but explanations for the observed differences are often absent or remain vague. The present study complements traditional approaches in help-seeking research by introducing a milieu approach, focusing on values and political attitudes as a possible explanation for differences in help-seeking for emotional mental health problems. METHODS: A representative cross-sectional survey of N = 3,042 respondents in Germany was conducted through face-to-face interviews about past help-seeking for mental health problems, socio-demographic characteristics and values and political attitudes. RESULTS: Multivariate logistic regression analyses indicated that belonging to a cosmopolitan intellectual milieu group was significantly associated with an increased likelihood of past help-seeking for mental health issues (psychotherapeutic/psychological help-seeking [OR = 2.09, 95% CI: 1.11-3.93, p < 0.05) and primary care (OR = 2.21, 95% CI: 1.15-4.24, p < 0.05]), whereas members of individualist and conservative milieu groups were less likely to report having sought help from a psychotherapist, but not from a general practitioner. Increased odds ratios were also found for a number of socio-demographic variables, such as being aged 26 years and over, a female gender and more than 12 years of formal education. Associations between socio-demographic variables remained significant, and the explained variance of the used models improved considerably when milieu variables were added. CONCLUSIONS: We discuss how milieu-specific patterns were relevant for explaining differences in mental health service use in addition to socio-demographic factors. It seems promising to consider help-seeking from a milieu perspective to improve disparities in access to and the use of psychotherapy as well as to resource allocation.

Direct-comb molecular spectroscopy with accurate, resolved comb teeth over 43 THz.

We demonstrate a dual-comb spectrometer using stabilized frequency combs spanning 177 to 220 THz (1360 to 1690 nm) in the near infrared. Comb-tooth-resolved measurements of amplitude and phase generate over 4×10(5) individually resolved spectral elements at 100 MHz point spacing and kilohertz-level resolution and accuracy. The signal-to-noise ratio is 100 to 3000 per comb tooth. Doppler-broadened phase and amplitude spectra of CO(2), CH(4), C(2)H(2), and H(2)O in a 30 m multipass cell agree with established spectral parameters, achieving high-resolution measurements with optical bandwidth generally associated with blackbody sources.

Precursor-product relationships between thymidine nucleotides and DNA in mammalian cells. II. Studies with dTDP and dTTP on cells partially lysed by Brij-58.

Cells from Chinese hamster ovary cell cultures were partially lysed with Brij-58 and incubated in a reaction mixture containing deoxyribonucleoside triphosphates and permitting semiconservative DNA replication. The reaction mixture was supplemented with [3H]dTTP and [14C]dTDP or, alternatively, with [14C]dTTP and [3H]dTDP, and at different times, 3H and 14C specific activities of dTTP and dTDP, as well as 3H/14C ratios in DNA were determined. Isotope ratios in DNA were in good agreement with those expected if dTTP but not dTDP was used as the proximate precursor for DNA synthesis. These data support the conclusion that the thymidine nucleotide used as proximate substrate for DNA replication is dTTP.

Comparative utilization of bromodeoxyuridine and iododeoxyuridine triphosphates for mammalian DNA replication in vitro.

Relative utilization, of dThd, BrdUrd, and IdUrd as exogenous precursors of DNA in vivo was compared with utilization of the respective triphosphates in vitro. To measure relative utilization, exponentially growing P-815 or CHO cells were incubated with different mixtures of dThd/BrdUrd or dThd/IdUrd in the presence of FdUrd and dCyd, and Brij-58 lysed cells with mixtures of dTTP/BrdUTP or dTTP/IdUTP. The degree of substitution of dTMP by BrdUMP or IdUMP in DNA newly synthesized in vivo or in vitro was determined by isopycnic centrifugation. BrdUrd was utilized by intact cells with equal efficiency as dThd, while IdUrd was less efficiently incorporated when the total concentration of dThd and the tested analogue was 3.10(-5) M. During semiconservative DNA replication in vitro, however, dTTP, BrdUTP and IdUTP were utilized with equal efficiency. Thus, we conclude that observed preference for dThd by intact cells incubated with dThd/IdUrd mixtures is related to characteristics of precursor metabolism and not to different affinities of the replication complex for the respective precursors. dUTP was rapidly dephosphorylated in vitro and no incorporation into DNA was detected.

Intrathymic transplantation of islet antigen affects CD8+ diabetogenic T-cells resulting in tolerance to autoimmune IDDM.

The acquisition of T-cell tolerance in the thymus is limited to those antigens expressed in the thymus at the time of T-cell development. Normally, islet antigens that are involved in insulin-dependent diabetes mellitus (IDDM) are not present in the thymus, but we have previously shown that transplantation of islets expressing relevant antigens into the thymus at the time of T-cell maturation results in prevention of IDDM in the multidose streptozotocin model of diabetes mellitus (MDSDM). Although both CD4+ and CD8+ T-cells are involved in the pathogenesis of this disease, the cells affected by intrathymic transplantation of islets are unknown. In this study, we have identified which T-cell subsets are affected by intrathymic islet antigens. Streptozotocin (STZ)-treated syngeneic islets were transplanted into the thymuses of C57BL/KsJ mice, and CD4+, CD8+, or both subsets of cells were transiently depleted with monoclonal antibodies (mAbs). After T-cell repopulation, animals that had received intrathymic islets followed by anti-CD8 mAb (P < 0.05) or both anti-CD4 and anti-CD8 mAbs (P < 0.01) but not anti-CD4 mAb alone were resistant to the development of autoimmune diabetes after five low doses of STZ. Insulitis was also reduced in mice receiving intrathymic islets and anti-CD8 (P < 0.025) or both anti-CD4 and anti-CD8 mAbs (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Invited Article: A compact optically coherent fiber frequency comb.

We describe the design, fabrication, and performance of a self-referenced, optically coherent frequency comb. The system robustness is derived from a combination of an optics package based on polarization-maintaining fiber, saturable absorbers for mode-locking, high signal-to-noise ratio (SNR) detection of the control signals, and digital feedback control for frequency stabilization. The output is phase-coherent over a 1-2 µm octave-spanning spectrum with a pulse repetition rate of ∼200 MHz and a residual pulse-to-pulse timing jitter <3 fs well within the requirements of most frequency-comb applications. Digital control enables phase coherent operation for over 90 h, critical for phase-sensitive applications such as timekeeping. We show that this phase-slip free operation follows the fundamental limit set by the SNR of the control signals. Performance metrics from three nearly identical combs are presented. This laptop-sized comb should enable a wide-range of applications beyond the laboratory.

Discovery and optimization of a novel class of orally active nonpeptidic endothelin-A receptor antagonists.

A novel class of endothelin-A receptor ligands was discovered by high-throughput screening. Lead structure optimization led to highly potent antagonists which can be synthesized in a short sequence. The compounds are endothelin-A-selective, are orally available, and show a long duration of action.

Determination of autofluorescence of red blood cells (RbCs) in uremic patients as a marker of oxidative damage.

AIM: It is suggested that the red blood cells (RBCs) of uremic patients have increased oxidative damage. The activities of different antioxidant enzymes and the levels of several antioxidants or lipid peroxidation products in RBCs are usually determined to estimate the oxidative stress in uremia. The autofluorescence of RBCs as measured by flow cytometry is caused by the formation of conjugated Schiff base compounds from aldehydes derived from lipid peroxidation and amino groups of phospholipids or cell proteins, and has been proposed as a marker of oxidative stress. The aim of this study was to evaluate if this method is suitable for estimation of oxidative stress in the RBCs of patients with different degrees of renal insufficiency. PATIENTS AND METHODS: To determine the oxidative damage in RBCs in uremia, the autofluorescence of RBCs was measured by flow cytometry in the following 3 groups of patients: group A: 16 patients with chronic renal failure (CRF); group B: 16 hemodialysis (HD) patients; group C: 16 patients with a well-functioning renal allograft. Twenty-four healthy volunteers served as controls. The basal value of RBC autofluorescence and the autofluorescence of RBCs after oxidative damage by treatment with 0.1 mM hydrogen peroxide (H2O2)/0.7 mM sodium azide were determined. RESULTS: In basal RBC autofluorescence values, no differences were found between the 3 groups and the controls. However, there was a significant correlation between the increase of serum creatinine and RBC autofluorescence in the group of patients with CRF (r = 0.521; p = 0.038). After H2O2 treatment, the RBC autofluorescence rose markedly in all individuals. This increase in RBC autofluorescence was significantly higher in the patients with CRF (p = 0.003) and in the HD patients (p = 0.001) compared to the controls. In contrast, there was no difference in RBC autofluorescence between the patients with renal allograft and the controls after H2O2 treatment. CONCLUSIONS: In conclusion, flow cytometry is a useful tool for determining oxidative damage in RBCs. The RBCs of uremic patients are more susceptible to oxidative damage induced by H2O2, likely caused by diminished antioxidant defense in the RBC membrane. Successful renal transplantation leads to a normal autofluorescence response in the RBCs after H2O2 treatment.

Colorimetric determination of low pH with Malachite Green.

A spectrophotometric method was developed for determination of concentration-based pH values from 0 to 2 with Malachite Green indicator. A quadratic model equation was based on the ratio of the absorbances of the peak at 618 nm and the isosbestic point at 518 nm. Normalization to the isosbestic point was used to stabilize the response because the color faded; the useful time interval was within 5 min after indicator addition. Model and verification sets agreed within +/-0.02 pH units between pH 0.3 and 1.8. This excellent precision makes the colorimetric method useful for acid determinations with a relative precision of > +/-5%. The presence of salts at a salt/acid equivalent ratio >0.1 caused a low pH bias.