Acid Usage Research Articles

Comparative Mitochondrial Genomes between the Genera Amiota and Phortica (Diptera: Drosophilidae) with Evolutionary Insights into D-Loop Sequence Variability.

To address the limited number of mitochondrial genomes (mitogenomes) in the subfamily Steganinae (Diptera: Drosophilidae), we assembled 12 complete mitogenomes for six representative species in the genus Amiota and six representative species in the genus Phortica. We performed a series of comparative and phylogenetic analyses for these 12 Steganinae mitogenomes, paying special attention to the commonalities and differences in the D-loop sequences. Primarily determined by the lengths of the D-loop regions, the sizes of the Amiota and Phortica mitogenomes ranged from 16,143-16,803 bp and 15,933-16,290 bp, respectively. Our results indicated that the sizes of genes and intergenic nucleotides (IGNs), codon usage and amino acid usage, compositional skewness levels, evolutionary rates of protein-coding genes (PCGs), and D-loop sequence variability all showed unambiguous genus-specific characteristics and provided novel insights into the evolutionary implications between and within Amiota and Phortica. Most of the consensus motifs were found downstream of the D-loop regions, and some of them showed distinct genus-specific patterns. In addition, the D-loop sequences were phylogenetically informative as the data sets of PCGs and/or rRNAs, especially within the genus Phortica.

Main Factors Shaping Amino Acid Usage Across Evolution.

The standard genetic code determines that in most species, including viruses, there are 20 amino acids that are coded by 61 codons, while the other three codons are stop triplets. Considering the whole proteome each species features its own amino acid frequencies, given the slow rate of change, closely related species display similar GC content and amino acids usage. In contrast, distantly related species display different amino acid frequencies. Furthermore, within certain multicellular species, as mammals, intragenomic differences in the usage of amino acids are evident. In this communication, we shall summarize some of the most prominent and well-established factors that determine the differences found in the amino acid usage, both across evolution and intragenomically.

Citric Acid Confers Broad Antibiotic Tolerance through Alteration of Bacterial Metabolism and Oxidative Stress.

Antibiotic tolerance has become an increasingly serious crisis that has seriously threatened global public health. However, little is known about the exogenous factors that can trigger the development of antibiotic tolerance, both in vivo and in vitro. Herein, we found that the addition of citric acid, which is used in many fields, obviously weakened the bactericidal activity of antibiotics against various bacterial pathogens. This mechanistic study shows that citric acid activated the glyoxylate cycle by inhibiting ATP production in bacteria, reduced cell respiration levels, and inhibited the bacterial tricarboxylic acid cycle (TCA cycle). In addition, citric acid reduced the oxidative stress ability of bacteria, which led to an imbalance in the bacterial oxidation-antioxidant system. These effects together induced the bacteria to produce antibiotic tolerance. Surprisingly, the addition of succinic acid and xanthine could reverse the antibiotic tolerance induced by citric acid in vitro and in animal infection models. In conclusion, these findings provide new insights into the potential risks of citric acid usage and the relationship between antibiotic tolerance and bacterial metabolism.

CITRIC ACID USAGE FOR INDUSTRIAL SAFETY INCREASE

The problem of mineral salt deposition is one of the most significant in the operation of oil fields. Salt deposits can lead to production downtime, an emergency situation and serious threat to personnel safety. To date, there are various methods of salt formation preventing. Chemical protection methods using inhibitors have become the most widespread. Despite the successes in the application of this type of protection, the accident rate remains at a high level. In this regard, the inhibitory protection effectiveness increasing remains relevant.The article suggests the use of citric acid (CA) as a co-inhibitor of salt deposits. The purpose of this work is to study the effectiveness of citric acid usage to prevent salt deposit formation and increase the level of industrial safety of oilfield equipment.This article describes a new method of combating salt deposits and presents the results of experimental studies to assess the citric acid effect on the salt deposition inhibitor (SDI) effectiveness. SDIs have been tested for their ability to prevent the salt formation. The ISO effectiveness was determined by the trilonometric method. It has been shown that one of the inhibitors in the dosage range of 30–100 g/t has extremely low protective properties, but with the citric acid addition, its effectiveness has increased. The best results of the protective effect of 75 % were achieved using citric acid and the second inhibitor.According to laboratory test results, a positive effect of citric acid on the inhibitor effectiveness was revealed. It has been experimentally proved that citric acid can be used to protect oilfield equipment from salt deposits.

Codon usage behavior distinguishes pathogenic Clostridium species from the non-pathogenic species

Genus Clostridium is of the largest genus in class Clostridia. It is comprised of spore-forming, anaerobic, gram-positive organisms. The members of this genus include human pathogens to free-living nitrogen fixing bacteria. In the present study, we have performed a comparison of the choice of preferred codons, codon usage patterns, dinucleotide and amino acid usage pattern of 76 species of Genus Clostridium. We found the pathogenic clostridium species to have smaller AT-rich genomes as compared to opportunistic and non-pathogenic clostridium species. The choice of preferred and optimal codons was also influenced by genomic GC/AT content of the respective clostridium species. The pathogenic clostridium species displayed a strict bias in the codon usage, employing 35 of the 61 codons encoding for 20 amino acids. Comparison of amino acid usage revealed an increased usage of amino acids with lower biosynthetic cost by pathogenic clostridium species as compared to opportunistic and non-pathogenic clostridium species. Smaller genome, strict codon usage bias and amino acid usage lead to lower protein energetic cost for the clostridial pathogens. Overall, we found the pathogenic members of genus Clostridium to prefer small, AT-rich codons to reduce biosynthetic costs and match the cellular environment of its AT-rich human host.

An update to tranexamic acid trends during the peripartum period in the United States, 2019 to 2021

Results from the 2017 World Maternal Antifibrinolytic trial found that patients who received tranexamic acid during delivery had significantly lower rates of death and hysterectomy. Several months after the World Maternal Antifibrinolytic trial publication, American College of Obstetricians and Gynecologists endorsed the consideration of tranexamic acid usage when traditional uterotonics fail during postpartum hemorrhage. Since then, tranexamic acid usage has become more mainstream for the treatment of postpartum hemorrhage. This study aimed to evaluate tranexamic acid trends in obstetrics both temporally and geographically within the United States. Additional outcomes included patient demographics and perinatal outcomes. This retrospective cohort study included 19 hospitals divided into East, Central, and West geographic regions within the Universal Health Services, Incorporated network. Rates of tranexamic acid use were compared from July 2019 through June 2021. Patient demographics and perinatal outcomes were analyzed for tranexamic acid recipients. During the two-year study period, 3.2% (1580/50,150) of patients received tranexamic acid during delivery. The western region of the United States demonstrated increased tranexamic acid use over the 2-year study period. Recipients of tranexamic acid were more likely to have a history of postpartum hemorrhage (P<.0001), chronic hypertension (P<.0001), preeclampsia (P<.0001), and/or diabetes (P=.004). Patients who received tranexamic acid did not have an increased likelihood of venous thromboembolism in comparison with those who did not receive tranexamic acid (8 [0.5%] vs 226 [0.5%]; P=.77). Of those who received tranexamic acid, 53.2% (840/1580) had an estimated blood loss <1000 mL. Nationally, a higher percentage of patients received tranexamic acid without a postpartum hemorrhage diagnosis compared with previous studies, and the western region of the United States had an overall increased use of tranexamic acid during delivery compared with previous years. There was no increased risk of venous thromboembolism in those who received tranexamic acid, regardless of postpartum hemorrhage diagnosis.

Microbial and Viral Genome and Proteome Nitrogen Demand Varies across Multiple Spatial Scales within a Marine Oxygen Minimum Zone.

Nutrient availability can significantly influence microbial genomic and proteomic streamlining, for example, by selecting for lower nitrogen to carbon ratios. Oligotrophic open ocean microbes have streamlined genomic nitrogen requirements relative to those of their counterparts in nutrient-rich coastal waters. However, steep gradients in nutrient availability occur at meter-level, and even micron-level, spatial scales. It is unclear whether such gradients also structure genomic and proteomic stoichiometry. Focusing on the eastern tropical North Pacific oxygen minimum zone (OMZ), we use comparative metagenomics to examine how nitrogen availability shapes microbial and viral genome properties along the vertical gradient across the OMZ and between two size fractions, distinguishing free-living microbes versus particle-associated microbes. We find a substantial increase in the nitrogen content of encoded proteins in particle-associated over free-living bacteria and archaea across nitrogen availability regimes over depth. Within each size fraction, we find that bacterial and viral genomic nitrogen tends to increase with increasing nitrate concentrations with depth. In contrast to cellular genes, the nitrogen content of virus proteins does not differ between size fractions. We identified arginine as a key amino acid in the modulation of the C:N ratios of core genes for bacteria, archaea, and viruses. Functional analysis reveals that particle-associated bacterial metagenomes are enriched for genes that are involved in arginine metabolism and organic nitrogen compound catabolism. Our results are consistent with nitrogen streamlining in both cellular and viral genomes on spatial scales of meters to microns. These effects are similar in magnitude to those previously reported across scales of thousands of kilometers. IMPORTANCE The genomes of marine microbes can be shaped by nutrient cycles, with ocean-scale gradients in nitrogen availability being known to influence microbial amino acid usage. It is unclear, however, how genomic properties are shaped by nutrient changes over much smaller spatial scales, for example, along the vertical transition into oxygen minimum zones (OMZs) or from the exterior to the interior of detrital particles. Here, we measure protein nitrogen usage by marine bacteria, archaea, and viruses by using metagenomes from the nitracline of the eastern tropical North Pacific OMZ, including both particle-associated and nonassociated biomass. Our results show higher genomic and proteomic nitrogen content in particle-associated microbes and at depths with higher nitrogen availability for cellular and viral genomes. This discovery suggests that stoichiometry influences microbial and viral evolution across multiple scales, including the micrometer to millimeter scale associated with particle-associated versus free-living lifestyles.

Pre-conception folic acid intake and attention deficit and hyperactivity disorder in children

Objectives: Attention Deficit Hyperactivity Disorder (ADHD) is a common disorder in children. It is thought to occur due to the interaction of many genetic and environmental factors during early development. Many studies have been conducted to investigate the etiology of ADHD. Some have investigated neurobiology, and others have investigated malnutrition and trace element deficiency. To investigate the effect of folic acid intake during the pre-conception period in terms of ADHD development. Methods: Five hundred and ninety-eight participants, 246 children with ADHD, and 352 healthy controls were enrolled. A questionnaire was completed, including socio-demographic information and the use of folic acid and other supplements such as a multivitamin, iron, and omega 3 during pregnancy. Data were examined to determine associations with ADHD. Mann-Whitney U tests for continuous variables, Pearson’s chi-square, and Fisher’s exact tests for categorical variables were used to compare groups. Results: The use of folic acid during pre-pregnancy among mothers who had a child with ADHD was 13% and this was 31% in the control group (p &amp;lt; 0.001). There was no statistically significant difference between the groups’ use of folic acid in pregnancy (p = 0.617). Other situations related to ADHD were advanced maternal age (p &amp;lt; 0.001 for both groups), abnormal double screening test results in pregnancy (27% vs. 5%, p &amp;lt; 0.001) and omega 3 use in pregnancy (p &amp;lt; 0.001). Conclusions: The use of folic acid before and during pregnancy is important for neurological development. However, there is little data on use before pregnancy in the literature. This study shows that folic acid taken before pregnancy may prevent ADHD in childhood. The present study recommends folic acid usage in planned pregnancy to prevent ADHD in the child.

PGRMC1 Ablation Protects from Energy-Starved Heart Failure by Promoting Fatty Acid/Pyruvate Oxidation.

Heart failure (HF) is an emerging epidemic with a high mortality rate. Apart from conventional treatment methods, such as surgery or use of vasodilation drugs, metabolic therapy has been suggested as a new therapeutic strategy. The heart relies on fatty acid oxidation and glucose (pyruvate) oxidation for ATP-mediated contractility; the former meets most of the energy requirement, but the latter is more efficient. Inhibition of fatty acid oxidation leads to the induction of pyruvate oxidation and provides cardioprotection to failing energy-starved hearts. One of the non-canonical types of sex hormone receptors, progesterone receptor membrane component 1 (Pgrmc1), is a non-genomic progesterone receptor associated with reproduction and fertility. Recent studies revealed that Pgrmc1 regulates glucose and fatty acid synthesis. Notably, Pgrmc1 has also been associated with diabetic cardiomyopathy, as it reduces lipid-mediated toxicity and delays cardiac injury. However, the mechanism by which Pgrmc1 influences the energy-starved failing heart remains unknown. In this study, we found that loss of Pgrmc1 inhibited glycolysis and increased fatty acid/pyruvate oxidation, which is directly associated with ATP production, in starved hearts. Loss of Pgrmc1 during starvation activated the phosphorylation of AMP-activated protein kinase, which induced cardiac ATP production. Pgrmc1 loss increased the cellular respiration of cardiomyocytes under low-glucose conditions. In isoproterenol-induced cardiac injury, Pgrmc1 knockout resulted in less fibrosis and low heart failure marker expression. In summary, our results revealed that Pgrmc1 ablation in energy-deficit conditions increases fatty acid/pyruvate oxidation to protect against cardiac damage via energy starvation. Moreover, Pgrmc1 may be a regulator of cardiac metabolism that switches the dominance of glucose-fatty acid usage according to nutritional status and nutrient availability in the heart.

Targeting of Glucose Transport and the NAD Pathway in Neuroendocrine Tumor (NET) Cells Reveals New Treatment Options

(1) Background: the potency of drugs that interfere with glucose metabolism, i.e., glucose transporters (GLUT) and nicotinamide phosphoribosyltransferase (NAMPT) was analyzed in neuroendocrine tumor (NET, BON-1, and QPG-1 cells) and small cell lung cancer (SCLC, GLC-2, and GLC-36 cells) tumor cell lines. (2) Methods: the proliferation and survival rate of tumor cells was significantly affected by the GLUT-inhibitors fasentin and WZB1127, as well as by the NAMPT inhibitors GMX1778 and STF-31. (3) Results: none of the NET cell lines that were treated with NAMPT inhibitors could be rescued with nicotinic acid (usage of the Preiss-Handler salvage pathway), although NAPRT expression could be detected in two NET cell lines. We finally analyzed the specificity of GMX1778 and STF-31 in NET cells in glucose uptake experiments. As previously shown for STF-31 in a panel NET-excluding tumor cell lines, both drugs specifically inhibited glucose uptake at higher (50 μM), but not at lower (5 μM) concentrations. (4) Conclusions: our data suggest that GLUT and especially NAMPT inhibitors are potential candidates for the treatment of NET tumors.

Mitogenome of the leaf-footed bug Notobitus montanus (Hemiptera: Coreidae) and a phylogenetic analysis of Coreoidea.

Notobitus montanus Hsiao, 1963 is a major pest of bamboos. The mitogenome of N. montanus (ON052831) was decoded using next-generation sequencing. The mitogenome, with 42.26% A, 30.54% T, 16.54% C, and 10.65% G, is 16,209 bp in size. Codon usage analysis indicated that high frequently used codons used either A or T at the third position of the codon. Amino acid usage analysis showed that leucine 2, phenylalanine, isoleucine and tyrosine were the most abundant in 31 Coreoidea species. Thirteen protein-coding genes (PCGs) were evolving under purifying selection, nad5 and cox1 had the lowest and strongest purifying selection stress, respectively. Correlation analysis showed that evolutionary rate had positive correlation with A+T content. No tandem repeat was detected in the non-coding region of N. montanus. The phylogenetic tree showed that Alydidae and Coreidae were not monophyletic. However, the topology of phylogenetic trees, based on 13 PCGs, was in accordance with that of tree based on both mitochondrial and nuclear genes but not ultraconserved element loci or combination of 13 PCGs and two rRNAs. It seems that their relationships are complex, which need revaluation and revision. The mitogenomic information of N. montanus could shed light on the evolution of Coreoidea.

Evolution of codon and amino acid usage in bacterial protein toxins

Toxin proteins are secreted by most pathogens as an integral part of pathogenic mechanism(s). The toxins act by either damaging the host cell membrane (for example, pore-forming toxins and RTX toxins) or by modulation of important cellular pathways (for example, inhibition of protein translation by ribosome-inactivating proteins). The mechanism of action of these toxins provides the pathogen with strategies for adaptation in the unfavorable host environment. Though, secreted by different pathogenic species, the protein toxins seem to share common features that allow the protein to bind to specific molecules and enter the host cell. Earlier studies have suggested role of several events like horizontal gene transfer and insertion-deletion mutations in evolution of protein toxins. The present study involving 125 bacterial protein toxins secreted by 49 pathogenic bacteria focuses on the role and constraints of the bacterial genome on evolution of codon and amino acid usage in respective bacterial protein toxins. We compare the nucleotide composition, codon and dinucleotide usage trends between different classes of bacterial protein toxins and between individual toxins and the parent bacterial genome expressing the toxin(s)

Mega-scale analysis of protein folding stability in biology and protein design.

Progress in DNA sequencing and machine learning have illuminated protein sequences and structures on an enormous scale, but protein thermodynamics remains invisible and largely unknown. New approaches are needed to reveal protein folding thermodynamics on the same enormous scale to overcome challenges in biology, medicine, and engineering. We present cDNA display proteolysis, a new method for measuring protein folding stability for up to 900,000 protein domains in a one-week experiment. From 1.7 million measurements in total, we curated a set of ∼800,000 high-quality folding stabilities comprehensively examining single amino acid variants of ∼350 natural and ∼250 de novo designed protein domains 40-72 amino acids in length. Using this immense dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also demonstrated applications of our method and dataset to protein design. The unique scale, speed, and accuracy of cDNA display proteolysis hold the potential to reveal protein folding thermodynamics across the entire universe of protein domains.

Impact of phylogeny on structural contact inference from protein sequence data.

Local and global inference methods have been developed to infer structural contacts from multiple sequence alignments of homologous proteins. They rely on correlations in amino acid usage at contacting sites. Because homologous proteins share a common ancestry, their sequences also feature phylogenetic correlations, which can impair contact inference. We investigate this effect by generating controlled synthetic data from a minimal model where the importance of contacts and of phylogeny can be tuned. We demonstrate that global inference methods, specifically Potts models, are more resilient to phylogenetic correlations than local methods, based on covariance or mutual information. This holds whether or not phylogenetic corrections are used, and may explain the success of global methods. We analyse the roles of selection strength and of phylogenetic relatedness. We show that sites that mutate early in the phylogeny yield false positive contacts. We consider natural data and realistic synthetic data, and our findings generalize to these cases. Our results highlight the impact of phylogeny on contact prediction from protein sequences and illustrate the interplay between the rich structure of biological data and inference.

Determinants of associations between codon and amino acid usage patterns of microbial communities and the environment inferred based on a cross-biome metagenomic analysis

Codon and amino acid usage were associated with almost every aspect of microbial life. However, how the environment may impact the codon and amino acid choice of microbial communities at the habitat level is not clearly understood. Therefore, in this study, we analyzed codon and amino acid usage patterns of a large number of environmental samples collected from diverse ecological niches. Our results suggested that samples derived from similar environmental niches, in general, show overall similar codon and amino acid distribution as compared to samples from other habitats. To substantiate the relative impact of the environment, we considered several factors, such as their similarity in GC content, or in functional or taxonomic abundance. Our analysis demonstrated that none of these factors can fully explain the trends that we observed at the codon or amino acid level implying a direct environmental influence on them. Further, our analysis demonstrated different levels of selection on codon bias in different microbial communities with the highest bias in host-associated environments such as the digestive system or oral samples and the lowest level of selection in soil and water samples. Considering a large number of metagenomic samples here we showed that microorganisms collected from similar environmental backgrounds exhibit similar patterns of codon and amino acid usage irrespective of the location or time from where the samples were collected. Thus our study suggested a direct impact of the environment on codon and amino usage of microorganisms that cannot be explained considering the influence of other factors.

Codon optimality has minimal effect on determining translation efficiency in mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a slow-growing, intracellular pathogen that exhibits a high GC-rich genome. Several factors, including the GC content of the genome, influence the evolution of specific codon usage biases in genomes. As a result, the Mtb genome exhibits strong biases for amino acid usage and codon usage. Codon usage of mRNAs affects several aspects of translation, including accuracy, efficiency, and protein folding. Here we address the effect of codon usage biases in determining the translation efficiency of mRNAs in Mtb. Unlike most commonly studied organisms, Mtb carries a single copy of each tRNA gene. However, we show that the relative levels of tRNAs in the Mtb tRNA pool vary by an order of magnitude. Our results show that the codons decoded by the abundant tRNAs indeed show higher adaptability. Moreover, there is a general positive correlation between genomic codon usage and the tRNA adaptability of codons (TAc). We further estimated the optimality of the codon and mRNAs by considering both the TAc and the tRNA demand. These measures did not show any correlation with mRNA abundance and translation efficiency. There was no correlation between tRNA adaptability and ribosome pausing as well. Taken together, we conclude that the translation machinery, and the tRNA pool of an organism, co-evolve with the codon usage to optimize the translation efficiency of an organism. Thus the deleterious effect of maladapted codons is not pronounced.

The compliance of free folic acid supplements among pregnant women in rural areas of Northwestern China: The role of related knowledge.

High prevalence of neural tube defects remains one of the major threats to newborns in rural China. Folic acid supplementation before and during early pregnancy can effectively reduce the risk of neural tube defects. Despite the efforts of the free folic acid mass distribution, the actual usage of folic acid supplements was still suboptimal among rural pregnant women in China. The objective of this study is to investigate if and how knowledge can influence the picking up and intake of the free folic acid supplements distributed by the government. We collected survey data from 821 pregnant women in rural areas of Shaanxi, China, in March and December of 2021. Face-to-face interviews and questionnaire surveys were conducted with every participant. Univariate and multivariate logistic regression analyses were performed to test the relationship between knowledge and dependent variables. Our study found that there were 76.4% of pregnant women would pick up folic acid supplements distributed by the government and only 44.5% of women would use folic acid before current pregnancy. Awareness of folic acid policy both affects the picking up (OR: 6.708, 95% CI: 4.672-9.632) and periconceptional intake (OR: 1.912, 95% CI:1.326-2.758) of folic acid supplements. Knowledge of health and nutrition in pregnancy showed no significant relationship with the picking up and periconceptional intake of folic acid supplements but was positively associated with the intake duration (Coefficient: 9.278, 95% CI: 2.966-15.591). Despite the relatively high level of picking up, the actual folic acid usage was not ideal among pregnant women in rural areas of China. Folic acid policy awareness was positively associated with the picking up and intake of folic acid before and during conception. Knowledge of health and nutrition about pregnancy was related to a longer duration of folic acid intake but had no impact on the picking up rate and periconceptional intake of folic acid supplements.

ВЛИЯНИЕ ОХЛАЖДЕНИЯ ТУШЕК В РАСТВОРАХ НАДУКСУСНОЙ КИСЛОТЫ НА ПИЩЕВУЮ ЦЕННОСТЬ МЯСА ЦЫПЛЯТ-БРОЙЛЕРОВ

The paper is devoted to studying the effect of broiler carcasses chilling in technological aid agent solutions at the base of peracetic acid on broiler meat nutritional value and amino acid composition. Meat nutritional value has been determined by standard methods usage/ Amino acid composition has been studied chromatografically with Sykam S-433-DS amino acid analyzer. The research has been shown that peracetic acid solutions (0.05–0.07% peracetic acid) usage for carcasses chilling (during 25 minutes) does not influence negatively on broiler meat nutrition and biologic value. Sensory meat quality doesn’t change excluding lighter surface color.

Recent advancements in design of nucleic acid nanocarriers for controlled drug delivery.

Research of nanoscale nucleic acid carriers has garnered attention in recent years due to their distinctive and controllable properties. However, current knowledge is limited in how we can efficiently utilize these systems for clinical applications. Several researchers have pioneered new and innovative nanocarrier drug delivery systems, but understanding physiochemical properties and behavior in vivo is vital to implementing them as clinical drug delivery platforms. In this review, we outline the most significant innovations in the synthesis, physical properties, and utilization of nucleic acid nanocarriers in the past 5 years, addressing the crucial properties which improve nanocarrier characteristics, delivery, and drug release. The challenges of controlling the transport of nucleic acid nanocarriers and therapeutic release for biological applications are outlined. Barriers which inhibit effective transport into tissue are discussed with emphasis on the modifications needed to overcome such obstacles. The novel strategies discussed in this work summarize the pivotal features of modern nucleic nanocarriers and postulate where future developments could revolutionize the translation of these tools into a clinical setting.

Analysis of hyaluronic acid usage in Municipality of Stip

Analysis of hyaluronic acid usage in Municipality of Stip