A genome-wide association study to investigate genetic loci associated with primary glaucoma in American Cocker Spaniels.

OBJECTIVE: To identify genetic associations with primary glaucoma (PG) in American Cocker Spaniels using a genome-wide association study (GWAS). ANIMALS: A nationwide ambidirectional case-control cohort study was performed in American Cocker Spaniels that had an ophthalmic examination performed by a veterinarian. Ninety-four dogs with PG (cases) and 111 dogs without glaucoma (controls) met phenotypic criteria and had a blood sample collected after receiving informed owner consent. PROCEDURES: Genomic DNA was extracted from whole blood samples and genotyped (CanineHD BeadChip, Illumina Inc). A case-control GWAS using a linear mixed model was performed, and 3 significance thresholds were calculated (1) using a Bonferroni correction on all single nucleotide polymorphisms (SNPs) included in the GWAS, (2) using a Bonferroni correction on only the unlinked SNPs from a pruned data set, and (3) using 10,000 random phenotype permutations. RESULTS: Following genotype data quality control, 89 cases and 93 controls were included in the GWAS. We identified an association on canine chromosome (CFA10); however, it did not reach statistical significance. Potential candidate genes within the surrounding linkage disequilibrium interval include coiled-coil domain containing 85A (CCDC85A) and extracellular growth factor containing fibulin extracellular matrix protein 1 (EFEMP1). CLINICAL RELEVANCE: Primary glaucoma in the American Cocker Spaniel is a complex heterogeneous disease that may be influenced by a locus on CFA10. The candidate genes CCDC85A and EFEMP1 within the identified linkage disequilibrium interval have been shown to be involved in human open-angle glaucoma.

Ig Enhancers Increase RNA Polymerase II Stalling at Somatic Hypermutation Target Sequences.

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

Healthcare Providers' Views on Hepatitis C Testing and Counseling Among Sexual Partners of Hepatitis-C-Infected Persons: An Online Survey.

BACKGROUND: Current hepatitis C virus (HCV) counseling guidelines do not recommend that HCV-infected patients notify their partners or encourage them to get tested. We aimed to assess healthcare professionals' knowledge of and attitudes toward counseling and testing recommendations for HCV-infected patients. METHODS: A 15-question, anonymous survey was designed and distributed via email to a convenience sample of healthcare professionals who work with Brown University or Boston University affiliated hospitals to assess their knowledge of and attitudes toward counseling recommendations for HCV-infected patients. The data was collected electronically and analyzed using descriptive statistical methods. RESULTS: Of the 55 respondents (a 20% response rate), 73% incorrectly believed that, at the time the survey was completed, CDC HCV testing guidelines already recommended partners of HCV-infected patients be tested for HCV infection. Furthermore, 80% of respondents believed recommendations should be revisited to explicitly include that HCV-infected patients encourage their partners to get tested. When counseling patients with HCV, 44% of respondents reported they always ask whether the patient's partners have been tested for HCV and 42% reported they sometimes do. Similarly, 42% reported they always suggest that the HCV-infected patient's partners be tested for HCV. CONCLUSIONS: Our survey shows that healthcare providers believe that HCV-counseling and testing recommendations could be revisited, with specific attention given to the promotion of HCV testing for partners of HCV-infected patients.

The use of hyaluronic acid subdermal filler for entropion in canines and felines: 40 cases.

PURPOSE: To evaluate the use of hyaluronic acid (HA) subdermal filler in canines and felines for entropion. METHODS: Complete ophthalmologic examination was performed by a board-certified veterinary ophthalmologist or ABVO-approved resident. Each case was characterized as primary, secondary, spastic or cicatricial entropion. HA subdermal filler, specifically Restylane® and Restylane Silk® , were utilized in dogs and cats, respectively. Subdermal injection was performed 1-2 mm from the eyelid margin in the affected area until normal eyelid conformation was achieved. All patients did not require sedation or general anesthesia. RESULTS: Forty animals (28 dogs and 12 cats) were included in the study. No local reaction to the HA dermal filler or any other complications other than minor skin bleeding at the injection sites were noted in all patients. Resolution of entropion and secondary complications including corneal ulceration, epiphora, and blepharospasm were noted by the first week after injection in the majority of cases. Three canines and one feline case failed to resolve the entropion, necessitating additional permanent surgical intervention. Two cases were submitted for histopathological analysis. Median follow-up time for all cases was 152.5 days (mean: 194.6 ± 142.7 days; range 9-419 days). Five patients died or were euthanized during the study for unrelated causes. CONCLUSIONS: Hyaluronic acid (HA) subdermal filler appears to be a safe, easy, reliable method for mild to moderate eyelid entropion not requiring general anesthesia. This procedure may be especially appropriate for geriatric patients and those with high anesthetic risk with entropion.

BACE1-cleavage of Sez6 and Sez6L is elevated in Niemann-Pick type C disease mouse brains.

It is intriguing that a rare, inherited lysosomal storage disorder Niemann-Pick type C (NPC) shares similarities with Alzheimer's disease (AD). We have previously reported an enhanced processing of ß-amyloid precursor protein (APP) by ß-secretase (BACE1), a key enzyme in the pathogenesis of AD, in NPC1-null cells. In this work, we characterized regional and temporal expression and processing of the recently identified BACE1 substrates seizure protein 6 (Sez6) and seizure 6-like protein (Sez6L), and APP, in NPC1-/- (NPC1) and NPC1+/+ (wt) mouse brains. We analysed 4-weeks old brains to detect the earliest changes associated with NPC, and 10-weeks of age to identify changes at terminal disease stage. Sez6 and Sez6L were selected due to their predominant cleavage by BACE1, and their potential role in synaptic function that may contribute to presentation of seizures and/or motor impairments in NPC patients. While an enhanced BACE1-cleavage of all three substrates was detected in NPC1 vs. wt-mouse brains at 4-weeks of age, at 10-weeks increased proteolysis by BACE1 was observed for Sez6L in the cortex, hippocampus and cerebellum of NPC1-mice. Interestingly, both APP and Sez6L were found to be expressed in Purkinje neurons and their immunostaining was lost upon Purkinje cell neurodegeneration in 10-weeks old NPC1 mice. Furthermore, in NPC1- vs. wt-mouse primary cortical neurons, both Sez6 and Sez6L showed increased punctuate staining within the endolysosomal pathway as well as increased Sez6L and BACE1-positive puncta. This indicates that a trafficking defect within the endolysosomal pathway may play a key role in enhanced BACE1-proteolysis in NPC disease. Overall, our findings suggest that enhanced proteolysis by BACE1 could be a part of NPC disease pathogenesis. Understanding the basic biology of BACE1 and the functional impact of cleavage of its substrates is important to better evaluate the therapeutic potential of BACE1 against AD and, possibly, NPC disease.

Comparing human peritoneal fluid and phosphate-buffered saline for drug delivery: do we need bio-relevant media?

An understanding of biological fluids at the site of administration is important to predict the fate of drug delivery systems in vivo. Little is known about peritoneal fluid; therefore, we have investigated this biological fluid and compared it to phosphate-buffered saline, a synthetic media commonly used for in vitro evaluation of intraperitoneal drug delivery systems. Human peritoneal fluid samples were analysed for electrolyte, protein and lipid levels. In addition, physicochemical properties were measured alongside rheological parameters. Significant inter-patient variations were observed with regard to pH (p < 0.001), buffer capacity (p < 0.05), osmolality (p < 0.001) and surface tension (p < 0.05). All the investigated physicochemical properties of peritoneal fluid differed from phosphate-buffered saline (p < 0.001). Rheological examination of peritoneal fluid demonstrated non-Newtonian shear thinning behaviour and predominantly exhibited the characteristics of an entangled network. Inter-patient and inter-day variability in the viscosity of peritoneal fluid was observed. The solubility of the local anaesthetic lidocaine in peritoneal fluid was significantly higher (p < 0.05) when compared to phosphate-buffered saline. Interestingly, the dissolution rate of lidocaine was not significantly different between the synthetic and biological media. Importantly, and with relevance to intraperitoneal drug delivery systems, the sustained release of lidocaine from a thermosensitive gel formulation occurred at a significantly faster rate into peritoneal fluid. Collectively, these data demonstrate the variation between commonly used synthetic media and human peritoneal fluid. The differences in drug release rates observed illustrate the need for bio-relevant media, which ultimately would improve in vitro-in vivo correlation.

Determination of Schirmer tear test-1 values in clinically normal alpacas (Vicugña pacos) in North America.

PURPOSE: To determine the normal reference range for Schirmer tear test-1 (STT-1) values in eyes of healthy alpacas (Vicugña pacos). METHODS: Complete ophthalmic examinations were performed on forty healthy alpacas (80 eyes). STT-1 values were obtained in both eyes of all alpacas using a commercial STT strip of a single lot number. Data were analyzed, using a doubly repeated measures ANOVA design, Student's paired t-test, and Pearson correlation test, with P ≤ 0.05 considered significant. RESULTS: The STT-1 values ± standard deviation (SD) were 20.80 ± 4.01 mm/min OD, 20.00 ± 4.13 mm/min OS, and 20.88 ± 4.04 mm/min OU (range 15.50-30.50 mm/min). No significant differences in STT-1 were found between OD and OS. STT-1 was not significantly affected by breed. Schirmer tear test-1 values were significantly increased by 3.45 mm/min for every 10 year increase in age. CONCLUSIONS: This study provides a reference range of STT-1 in the healthy alpaca which can assist veterinarians in diagnosing potential keratoconjunctivitis sicca, tear film abnormalities, as well as ocular surface diseases in alpacas.

Use of equine amniotic membrane free-island grafts for treatment of a midstromal corneal ulcer and descemetocele in a snow leopard (Panthera uncia).

CASE DESCRIPTION A 7-year-old sexually intact female snow leopard (Panthera uncia) was examined because of blepharospasm, periocular discharge, ventral deviation of the upper eyelid cilia, third eyelid prolapse, and corneal opacity of the right eye. CLINICAL FINDINGS An ophthalmic examination performed with the patient anesthetized revealed a 3 × 3-mm ulcer that extended approximately 60% of the depth of the right cornea and was accompanied by perilesional and intralesional cellular infiltrates and active vascularization. The upper eyelid of the right eye also had a previously repaired coloboma resulting in trichiasis. TREATMENT AND OUTCOME Surgical intervention was elected after 5 weeks of medical management including topical administration of autologous serum and topical, subconjunctival, and systemic administration of antimicrobials failed to yield any improvement in the ulcer. Equine amniotic membrane free-island graft placement and eyelid revision surgeries were performed. Two and a half weeks later, a descemetocele was diagnosed ventrolateral to the original ulcer, and a second equine amniotic membrane free-island grafting procedure was performed. Both grafts healed without further intervention. CLINICAL RELEVANCE Equine amniotic membrane free-island grafts were used to successfully repair a corneal ulcer and descemetocele in a snow leopard. The grafting procedure spared the affected globe and resulted in satisfactory cosmesis and functional vision. This procedure should be considered as an option for corneal repair in nondomestic species for which postoperative care and medical treatment options are limited.

Sexual health, risky sexual behavior and condom use among adolescents young adults and older adults in Chiang Mai, Thailand: findings from a population based survey.

BACKGROUND: Sexual health is one of the key dimensions of health across all ages. Understanding risky sexual behaviors remains an important area of public health research. This study aimed to explore sexual health, risky sexual behaviors and factors associated with recent condom use as condom use is considered a main intervention proven to reduce negative health consequences of risky sexual behaviors, specifically related to sexually transmitted infections and unintended pregnancies. A stratified two-stage cluster sampling technique survey was conducted in Chiang Mai, Thailand. Information was obtained about age of first sexual intercourse, sexual activity, condom use, number of partners and history of drug/alcohol use prior to sexual activities within the past 3 months. A weighted analysis was performed to account for data clustering. RESULTS: It is estimated that most men (93%) and women (86%) in Chiang Mai have engaged in sexual intercourse. More than 70% of the people in Chiang Mai over age 30 remained sexually active in the past 3 months, even for populations over age 50. Eight percent of male teenagers reported having more than one sexual partner in the past 3 months. Regular condom use was reported in less than 5% of the population (6.6% men and 3.1% women). CONCLUSIONS: Our study demonstrated that sexual health is an important public health issue across all age groups. Condom use has been promoted as one way to minimize and prevent unintended consequences of sexual behavior but overall use remains low.

Orbital rhabdomyosarcoma and traumatic neuroma following enucleation for a uveal schwannoma in a dog: a case report.

A 4-year-old, female spayed Siberian husky with history of a uveal schwannoma presented for orbital swelling 9 months after enucleation. A second, malignant tumor developed in the same orbit. Therefore, uveal schwannomas may warrant early surgical intervention in the dog.

Comparison of intraocular pressure measurements using rebound (TonoVet® ) and applanation (TonoPen-XL® ) tonometry in clinically normal alpacas (Vicugña pacos).

PURPOSE: To evaluate intraocular pressure (IOP) estimates in eyes of healthy alpacas (Vicugña pacos) using rebound (TonoVet® ) in comparison with applanation (TonoPen-XL® ) tonometry. METHODS: Complete ophthalmic examinations were performed on forty healthy alpacas (80 eyes). IOP measurements using both TonoVet® and TonoPen-XL® tonometers were obtained OU. Data were analyzed, using a doubly repeated-measures anova design and Student's paired t-test, with P ≤ 0.05 considered significant. RESULTS: The mean IOP values ±SD via rebound tonometry were 14.20 mm ± 2.58 mm OD, 14.22 mm ± 2.90 mm OS, and 14.21 ± 2.73 mmHg OU (range 8.67-20.67 mmHg). The IOP values ±SD via applanation tonometry were 12.49 ± 2.81 mmHg OD, 12.53 ± 2.79 mmHg OS, and 12.51 ± 2.78 mmHg OU (range 6.00-19.33 mmHg). There was a significant difference (P = 0.002) in the IOP obtained between the tonometers, with the rebound tonometer having a 1.7 mmHg (0.69-2.71 mmHg, 95% confidence interval (CI)) higher IOP compared to the applanation tonometer. No significant differences in IOP were found between OD and OS. Age, gender, and breed did not significantly affect IOP values. CONCLUSIONS: IOP readings from the rebound tonometer were statistically higher than those from the applanation tonometer; however, this is not considered clinically significant. The accuracy of rebound tonometry in diseased alpaca eyes remains to be determined.

Effect of topical latanoprost 0.005% on intraocular pressure and pupil diameter in normal and glaucomatous cats.

OBJECTIVE: To determine the effects of latanoprost on intraocular pressure (IOP) and pupil diameter (PD) in cats with inherited primary congenital glaucoma (PCG) and normal cats. ANIMALS STUDIED AND PROCEDURES: IOP and PD were measured in both eyes (OU) of 12 adult cats (six normal, six PCG), three times per week for 3 weeks prior to, for 3 weeks during, and for 2 weeks following twice-daily treatment with 0.005% latanoprost to the right eye (OD) and vehicle to the left (control) eye (OS). IOP and PD were measured hourly, for 8 h, 1 day prior to, and on the first and last days of treatment. Aqueous humor flow rate (AHF) was determined at baseline and at the end of the treatment phase in six normal cats. RESULTS: Mean IOP was significantly lower in treated vs. control eyes of PCG cats, for up to 8 h following a single latanoprost treatment, and a maximal IOP reduction of 63% occurred in treated eyes at 3 h. Latanoprost acutely lowered IOP in cats with PCG, but this effect appeared to diminish over 3 weeks of treatment. AHF was modestly increased in the treated eyes of normal cats after 3 weeks of latanoprost treatment, although IOP was not significantly affected. Latanoprost caused miosis, with rebound mydriasis at 24 h posttreatment, in the treated eyes of all cats. CONCLUSIONS: Further research is needed to determine the suitability and efficacy of latanoprost treatment for long-term IOP-lowering in cats with PCG or other forms of glaucoma.

Ventral transpalpebral anterior orbitotomy: surgical description and report of 3 cases.

PURPOSE: To describe a surgical approach to allow access to the ventral anterior canine orbit and report outcomes of three cases. SURGICAL TECHNIQUE: After induction of general anesthesia and aseptic preparation of the surgical site, a 2.5- to 3-cm curvilinear skin incision was created through the inferior eyelid at the level of the ventral orbital rim. A combination of sharp and blunt dissection facilitated entrance into the ventral anterior orbital space for the removal of diseased tissues or allows for drainage of purulent debris. Two-layer closure was performed, and postoperative lateral temporary tarsorrhaphy sutures were retained to provide globe protection. RESULTS: Three dogs underwent unilateral ventral transpalpebral anterior orbitotomy. Prior to surgery, apart from a complete ophthalmic examination, ocular ultrasound was used to diagnose orbital disease in two cases, and MRI was utilized in the third case. Exploratory orbitotomy revealed a large mucocele in case 1, orbital bacterial abscessation in case 2, and necrotizing zygomatic sialoadenitis in case 3. Clinical exophthalmos resolved immediately after surgery. The surgical site in all cases healed within 2 weeks. One patient had a superficial corneal ulceration 2 weeks after surgery which healed uneventfully. Recurrence of orbital disease was not noted in any case. CONCLUSIONS: Ventral transpalpebral anterior orbitotomy is a simple procedure that allows easy access to the ventral anterior orbit to allow for removal of diseased tissues or to facilitate drainage of abscessation. Recurrence of orbital disease was not seen in any patient, with one patient experiencing blindness as a long-term complication following the procedure.

Targeting of somatic hypermutation by immunoglobulin enhancer and enhancer-like sequences.

Somatic hypermutation (SH) generates point mutations within rearranged immunoglobulin (Ig) genes of activated B cells, providing genetic diversity for the affinity maturation of antibodies. SH requires the activation-induced cytidine deaminase (AID) protein and transcription of the mutation target sequence, but how the Ig gene specificity of mutations is achieved has remained elusive. We show here using a sensitive and carefully controlled assay that the Ig enhancers strongly activate SH in neighboring genes even though their stimulation of transcription is negligible. Mutations in certain E-box, NFκB, MEF2, or Ets family binding sites--known to be important for the transcriptional role of Ig enhancers--impair or abolish the activity. Full activation of SH typically requires a combination of multiple Ig enhancer and enhancer-like elements. The mechanism is evolutionarily conserved, as mammalian Ig lambda and Ig heavy chain intron enhancers efficiently stimulate hypermutation in chicken cells. Our results demonstrate a novel regulatory function for Ig enhancers, indicating that they either recruit AID or alter the accessibility of the nearby transcription units.

A critical context-dependent role for E boxes in the targeting of somatic hypermutation.

Secondary B cell repertoire diversification occurs by somatic hypermutation (SHM) in germinal centers following Ag stimulation. In SHM, activation-induced cytidine deaminase mutates the V region of the Ig genes to increase the affinity of Abs. Although SHM acts primarily at Ig loci, low levels of off-target mutation can result in oncogenic DNA damage, illustrating the importance of understanding SHM targeting mechanisms. A candidate targeting motif is the E box, a short DNA sequence (CANNTG) found abundantly in the genome and in many SHM target genes. Using a reporter assay in chicken DT40 B cells, we previously identified a 1928-bp portion of the chicken IgL locus capable of supporting robust SHM. In this article, we demonstrate that mutation of all 20 E boxes in this fragment reduces SHM targeting activity by 90%, and that mutation of subsets of E boxes reveals a functional hierarchy in which E boxes within "core" targeting regions are of greatest importance. Strikingly, when the sequence and spacing of the 20 E boxes are preserved but surrounding sequences are altered, SHM targeting activity is eliminated. Hence, although E boxes are vital SHM targeting elements, their function is completely dependent on their surrounding sequence context. These results suggest an intimate cooperation between E boxes and other sequence motifs in SHM targeting to Ig loci and perhaps also in restricting mistargeting to certain non-Ig loci.

Identification of core DNA elements that target somatic hypermutation.

Somatic hypermutation (SHM) diversifies the V region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high-affinity Abs are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). Whereas mistargeting of SHM and AID results in mutations and DNA damage in many non-Ig genes, they act preferentially at Ig loci. The mechanisms responsible for preferential targeting of SHM and AID activity to Ig loci are poorly understood. Using an assay involving an SHM reporter cassette inserted into the Ig L chain locus (IgL) of chicken DT40 B cells, we have identified a 1.9-kb DIVAC (diversification activator) element derived from chicken IgL that supports high levels of AID-dependent mutation activity. Systematic deletion analysis reveals that targeting activity is spread throughout much of the sequence and identifies two core regions that are particularly critical for function: a 200-bp region within the IgL enhancer, and a 350-bp 3' element. Chromatin immunoprecipitation experiments demonstrate that whereas DIVAC does not alter levels of several epigenetic marks in the mutation cassette, it does increase levels of serine-5 phosphorylated RNA polymerase II in the mutation target region, consistent with an effect on transcriptional elongation/pausing. We propose that multiple, dispersed DNA elements collaborate to recruit and activate the mutational machinery at Ig gene variable regions during SHM.

Forensic DNA analysis.

Before the routine use of DNA profiling, blood typing was an important forensic tool. However, blood typing was not very discriminating. For example, roughly 30% of the United States population has type A-positive blood. Therefore, if A-positive blood were found at a crime scene, it could have come from 30% of the population. DNA profiling has a much better ability for discrimination. Forensic laboratories no longer routinely determine blood type. If blood is found at a crime scene, DNA profiling is performed. From Jeffrey's discovery of DNA fingerprinting to the development of PCR of STRs to the formation of DNA databases, our knowledge of DNA and DNA profiling have expanded greatly. Also, the applications for which we use DNA profiling have increased. DNA profiling is not just used for criminal case work, but it has expanded to encompass paternity testing, disaster victim identification, monitoring bone marrow transplants, detecting fetal cells in a mother's blood, tracing human history, and a multitude of other areas. The future of DNA profiling looks expansive with the development of newer instrumentation and techniques.

The levels of water-soluble and triton-soluble Aß are increased in Alzheimer's disease brain.

Although plaques composed of the amyloid ß-protein (Aß) are considered a defining feature of Alzheimer's disease (AD), they are also found in cognitively normal individuals and extensive evidence suggests that non-plaque, water-soluble forms of Aß may play a role in AD pathogenesis. However, the relationship between the levels of water-soluble Aß and the clinical severity of disease has never been investigated. Here, we present results of a pilot study designed to examine the levels of water-soluble forms of Aß in brains of individuals who died at clinically distinct stages of AD. Using a serial extraction method, we also investigated the levels of triton-soluble and formic acid-soluble Aß. We found that water-soluble and detergent-soluble Aß monomer and SDS-stable dimer were elevated in AD and that the levels of water soluble Aß did not increase with plaque pathology. These results support the notion that both water- and detergent-soluble Aß are important in AD and are not simply released from plaques by mechanical disruption. Moreover, the fact that the levels of water- and triton-soluble Aß were similar in very mild/mild AD and moderate/severe AD suggests that once a certain level of these species is attained, further accumulation is not necessary for the disease to progress. Consequently, therapeutic targeting of water-soluble Aß should best benefit individuals in earliest phases of the disease process.

Isolation of low-n amyloid ß-protein oligomers from cultured cells, CSF, and brain.

Recent data suggest that soluble, non-fibrillar assemblies of the amyloid ß-protein (Aß) may mediate the synaptic deficits that characterize the early stages of Alzheimer's disease. Consequently, much effort has been expended in isolating and studying a variety of different Aß assemblies. Here, we describe the use of immunoprecipitation/western blotting and size exclusion chromatography/western blotting to characterize Aß present in conditioned medium from cultured cells, human cerebrospinal fluid, and human cortex extracted with aqueous buffer, detergent, and formic acid.