Days Of Incubation Research Articles

Control of inorganic and organic phosphorus molecules on microbial activity, and the stoichiometry of nutrient cycling in soils in an arid, agricultural ecosystem

Background The dynamics of carbon (C), nitrogen (N), and phosphorus (P) in soils determine their fertility and crop growth in agroecosystems. These dynamics depend on microbial metabolism, which in turn depends on nutrient availability. Farmers typically apply either mineral or organic fertilizers to increase the availability of nutrients in soils. Phosphorus, which usually limits plant growth, is one of the most applied nutrients. Our knowledge is limited regarding how different forms of P impact the ability of microbes in soils to produce the enzymes required to release nutrients, such as C, N and P from different substrates. Methods In this study, we used the arable layer of a calcareous soil obtained from an alfalfa cropland in Cuatro Cienegas, México, to perform an incubation experiment, where five different phosphate molecules were added as treatments substrates: three organic molecules (RNA, adenine monophosphate (AMP) and phytate) and two inorganic molecules (calcium phosphate and ammonium phosphate). Controls did not receive added phosphorus. We measured nutrient dynamics and soil microbial activity after 19 days of incubation. Results Different P molecules affected potential microbial C mineralization (CO2-C) and enzyme activities, specifically in the organic treatments. P remained immobilized in the microbial biomass (Pmic) regardless of the source of P, suggesting that soil microorganisms were limited by phosphorus. Higher mineralization rates in soil amended with organic P compounds depleted dissolved organic carbon and increased nitrification. The C:N:P stoichiometry of the microbial biomass implied a change in the microbial community which affected the carbon use efficiency (CUE), threshold elemental ratio (TER), and homeostasis. Conclusion Different organic and inorganic sources of P affect soil microbial community structure and metabolism. This modifies the dynamics of soil C, N and P. These results highlight the importance of considering the composition of organic matter and phosphate compounds used in agriculture since their impact on the microbial activity of the soil can also affect plant productivity.

Enhanced Foliar Litter Decomposition Rate of Pinus massoniana When Admixed with Broadleaf Species

In the global ecosystem, the slow decomposition of coniferous forest litter has caused a number of ecological problems, among which is the decay of China’s Pinus massoniana litter. It has been pointed out that converting pure P. massoniana plantations into mixed forests with broadleaf species can improve ecosystem services. Therefore, the selection of mixed species is key for the success or failure of the conversion of near-natural forests. In this study, from the perspective of apoplastic decomposition, the leaf litter of P. massoniana was mixed with three common native broadleaf species, namely Choerospondias axillaries, Cinnamomum camphora, and Cyclobalanopsis glauca, using an indoor incubation method to systematically analyse the differences in the decomposition rates of apoplastic material in each mixture, and to provide a theoretical basis for the selection and mixing of tree species for the management of near-natural forests in P. massoniana forests. After 175 days of indoor incubation of the foliar litter under dark conditions at 25 °C, the residual dry matter of the mixed apoplastic litter of P. massoniana and the three broadleaf trees was lower than that of P. massoniana. It indicated that the incorporation of broadleaf apoplastic foliage promoted litter decomposition, with the most pronounced effect in the case of admixture with C. Camphora. Compared with the group of pure P. massoniana alone, the remaining mass and residual rate decreased by 0.56 g and 9.45%, respectively. The regression equation of Olson’s negative exponential decay model showed that the P. massoniana + C. Camphora mixture had the fastest decomposition rate (k) of 1.305, an increase of 0.237, a decrease in half-life of 0.11 years, and a decrease in turnover period of 0.49 year, compared to the P. massoniana alone group. Most of the measured values throughout the incubation period were significantly lower than the predicted values, suggesting that there was a non-additive and synergistic effect of litter mixing.

Scientific evaluation on biodegradable and biofunctional polyurethane incorporated chitosan/elastin electrospun membranes in compared with synthetic polymeric expanded polytetrafluoroethylene and polyethylene terephthalate vascular membranes

Cardiovascular disease is a life-threatening health problem. Employing vascular membranes is one of the treatment options in cardiovascular surgery to replace damaged vascular tissues. However, certain limitations associated with synthetic polymeric vascular membranes (expanded polytetrafluoroethylene (e-PTFE) and polyethylene terephthalate (PET)) such as low biodegradability and biofunctional, may lead to thrombosis, inflammatory response, and tissue necrosis. Polyurethane (PU) is commonly applied in vascular engineering, owing to its good biocompatibility and biodegradability. Incorporating chitosan (CS) and elastin (EL) into the PU matrix, specifically in the design of electrospun nanofibrous membrane, is expected to enhance the biological properties of PU. These improvements are crucial to overcome the limitations of synthetic vascular membranes. Previously, there was no scientific comparison between synthetic vascular membranes and the improved design of PU-based nanofibrous membranes. This scientific comparison is pivotal to allow more research exploration on biodegradable and biofunctional materials towards the fabrication of vascular membranes. Herein, the physicochemical, biodegradability, and biofunctional capability of the electrospun PU-based electrospun membranes and two synthetic membranes were compared. Both PU and PU/CS/ES electrospun membranes were annotated with N–H, C–H, and C–N groups with the appearance of elongated fibers. The –CF2 group was presented in the e-PTFE membranes with the view of expanded fibril structure and interconnecting nodes. Whereas for the PET membranes, C=C, C=O, and C–O groups were noticed with the appearance of homogeneous knitted morphology. The PU/CS/ES membranes were found to be more hydrophilic than the PU and the synthetic membranes. The incorporation of CS and EL in the matrix of electrospun PU has improved the PU's biodegradability up to 23.86 ± 5.86 % at 60 days of incubation with the view of swollen and degraded elongated fibers, following the degradation test. Both synthetic polymeric membranes were declared non-biodegradable with less than 7 % degradation at similar incubation timepoint. The PU/CS/ES membranes were also found biofunctional with the most 82 ± 0.49 % of cell viability, protruded cell filopodia, and 44.24 ± 13.99 % scratch cell closure within 24 h of incubation. The incorporation of CS and EL into the PU matrix has improved the biodegradability and biofunctionality compared to the pure PU and synthetic polymeric membranes. Thus, highlight the suitability of PU/CS/ES electrospun membranes to be applied in cardiovascular tissue engineering.

ALGINATE-BASED CELL ENCAPSULATION USING DIFFERENT CROSSLINKER ELEMENTS

Alginate microcapsules are the most frequently used materials for cell transplantation. Different crosslinkers affect crosslinking affinity, which has a significant influence on microcapsule properties. The objective was to prepare in vitro microcapsules using calcium, barium, iron, manganese, nickel, and strontium as divalent cations to observe their potential for use in cell transplantation. Sodium alginate was added dropwise to the individually prepared crosslinkers to observe diffusion-based gelling. Alginate microcapsules were investigated regarding capsule stability, physiological properties, and cell viability. After 30 days of incubation, cell viability was greater than 90% for the cell-encapsulated microcapsules when crosslinked with CaCl2 and NiCl2. Viability decreased in the following order: [Formula: see text]. A compression test was performed to investigate the required force to deform 30% of microcapsules, and only MnCl2, FeCl2 (180[Formula: see text]mM), and NiCl2 (50[Formula: see text]mM) demonstrated higher resistance to the applied force than CaCl2. Except for the FeCl2 group, all cell-encapsulated microcapsules remained intact for 45 days. Potential sensitivities to CaCl2 during cell transplantation may compel alternative crosslinker usage, and our study revealed that NiCl2 and BaCl2 can be used as alternative crosslinkers to CaCl2 due to their high cell viability and consistent stability.

Unlocking higher yields in Urochloa brizantha: the role of basalt powder in enhancing soil nutrient availability

This study investigates the effect of basalt powder on soil fertility through changes in its chemical characteristics and the response of Urochloa brizantha to different application doses. The experiment was conducted in a greenhouse using two distinct soil types: Oxisol (OX) and Typic Quartzipsamment (TQ). Basalt powder dosages were determined based on the calcium levels required to adjust calcium contents in each soil (Ca2+) to recommended levels for the U. brizantha,ranging from 0 to 4 times the recommended amount. After 170 days of incubation, soil samples were collected for analysis to evaluate soil attributes. U. brizantha was then planted to assess the efficiency of basalt powder, which was harvested at 40 days after sowing (first cut) and 70 days after sowing (second cut). The accumulation of nutrients and shoot dry mass (SDM) production were subsequently measured. The findings revealed that applying the highest dose of basalt powder (96 Mg ha−1) substantially elevated pH, and increased concentrations of phosphorus (P), potassium (K+), calcium (Ca2+), magnesium (Mg2+), copper (Cu), boron (B), and silicon (Si) in Oxisol (OX), while decreasing aluminum (Al3+) contents in the same soil. Furthermore, an average increase in the SDM of both U. brizantha harvests was observed, with a 6% increase in TQ and a 27% increase in OX. Additionally, an increase in the accumulation of P, K, Ca, Mg, and sulphur (S) in the shoot dry mass of the plants was observed with the basalt powder doses. These findings suggest that basalt powder has the potential to be an environmentally friendly alternative for soil fertilization, with positive impacts on soil chemical attributes and plant growth and nutrition.

Comparative Analysis of Ligninolytic Potential among Pleurotus ostreatus and Fusarium sp. with a Special Focus on Versatile Peroxidase

Lignocellulosic biomass is contemplated to be an inexpensive and copious feedstock that can be used for numerous industrial applications. However, lignin forms the lignin sheath and provides a physical barrier to enzymatic hydrolysis. In addition, lignin physically blocks cellulase, preventing it from being combined with the substrate in a process known as non-productive binding. Therefore, the depletion of lignin is a crucial method for obtaining fermentable sugars from the lignocellulosic biomass. Different white-rot fungi secrete different sets of lignin-mineralizing enzymes and each fungus secretes one or more of the three enzymes essential for lignin degradation. Among efficient redox enzymes, versatile peroxidase is extensively studied for its ability to degrade aromatics without the need for a mediator or polyvalent catalytic site. However, the presence of versatile peroxidase in F. spp. has not been studied. This study was planned with the objective of screening and comparing the production of versatile peroxidase enzymes from F. spp. and a standard culture of Pleurotus ostreatus MTCC-142. These fungal strains were first screened on solid media containing tannic acid, malachite green, or bromocresol green. The potency index for the tannic acid, malachite green, and bromocresol green on the 16th day of incubation was reported to be 1.28, 1.07, 1.09, and 1.10, respectively. Versatile peroxidase production patterns were investigated under solid state fermentation conditions for a period of 25 days at different temperatures ranging from 10 to 35 °C. The highest versatile peroxidase activity (592 UL−1) in F. sp. was observed at 30 °C after the 7th day of incubation. The molecular confirmation showed the presence of the vp gene in F. sp. along with Pleurotus ostreatus MTCC-142. The results determined that F. sp. possesses a versatile peroxidase enzyme and is able to degrade lignin efficiently, and thus it could be utilized as an alternative to other ligninolytic enzyme-producing fungi.

First report of Rhizoctonia blight caused by Rhizoctonia solani on Chinese cabbage (Brassica rapa subsp. chinensis) in India.

During January and February 2021, foliar blight symptoms were observed on the leaves of Chinese cabbage (Pak choi) at Lembucherra research farm, College of Agriculture, Tripura, India. The incidence of disease symptoms ranged from 5 to 10% of the plants observed in the field. The symptomatic leaves showed grayish colored water-soaked lesions with an irreguar shape and size. A total of 10 symptomatic leaves (1 leaf per plant) from Chinese cabbage infected plant were sampled, surface decontaminated with 1% NaOCl, washed twice in sterile water, plated on 2% water agar, and incubated at 25 ± 2°C. Hyphal tips from mycelium of 7-day old culture (2 isolates from two different plants) with right-angled branching were transferred to potato dextrose agar (PDA) media (SRL, India). Cream or light brown hyphae that branched at right angles, with septa near the point of the origin of hyphae, and a slight constriction at the base of the branch) were visible under a microscope. Olive-brown sclerotia were observed after 5 days of incubation. Multiple nuclei per cell were visible after staining with 4', 6-diamidino-2-phenylindole (Estandarte et al. 2016). Based on morphological characteristics (Parmeter et al. 1970) the isolates TP36 and TP37 were identified as Rhizoctonia solani. The internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were amplified with ITS1& ITS4 (White et al. 1990) and (GAPDH F-5'- CAAGGAGAACCCAGGTGTTAAG-3' and GAPDH R- 5'-GGCGTCGAAGATAGAAGAGTGT-3') respectively for both isolates and sequenced (accession #. PP458158, PP458159, PP425343, PP425344). BLASTn analysis showed 99.26%( 668/673 nt) to 99.46% (659/664 nt) identity with R. solani sequences (GenBank MG397062.1 and KX674524.1) for ITS and 98.42% (552/562 nt) to 100% 540/540 nt)identity with R. solani sequences (GenBank HQ425709.1 and CP102644.1) for GAPDH. Isolates TP36 and TP37 were deposited in the Indian Type Culture Collection (ITCC), New Delhi as R. solani (nos. 9154 and 9319, respectively). Both isolates were amplified using (anastomosis group) AG1 subgroup specific primers (Matsumoto 2002; Prashantha et al. 2021) to identify their AG. The presence of a 265 bp amplicon for both isolates suggested that they belong to AG1-IA. A multilocus analysis of R. solani isolates from different host plants with concatenated sequences ITS and GAPH showed that TP36 and TP37 are closely related to rice isolate RS107. A pathogenicity test on five plants per treatment was conducted and repeated twice on one month old Chinese cabbage plants (hybrid, TOKITA, India) grown under glasshouse conditions in a sterilized mixture of soil and sand (3:1) at 27-28oC during January 2024 at ICAR-IARI, New Delhi. R. solani isolates TP36 and TP37 were grown on PDA and plants were inoculated by placing single sclerotia of 10-day old colony on different plant parts and covering it with moist cotton. After 7 day, typical lesions of R. solani infection were visible. No symptoms were observed on the control plants. The fungus was reisolated from the inoculated plants and identified as R. solani based on morphology. R. solani has previously been reported to cause disease on some members of Brassicaceae in different countries (Budge et al. 2009; Hua et al. 2014). Based on literature available this is the first report of R. solani infecting Chinese cabbage in India.

Extracts and Terpenoids from Stevia Species as Potential Anthelmintics for Neglected Tropical Diseases Caused by Cestode Parasites.

Cestodes are etiological agents of neglected diseases such as echinococcosis and cysticercosis, which are major public health problems. Antiparasitic treatment relies on a small number of approved drugs, which are often only partially effective, poorly tolerated and require prolonged administration. Thus, the discovery of novel potential treatments is critical. The Stevia genus (Asteraceae) includes species that are recognized as a source of bioactive compounds, with many species associated with medicinal uses. In this study, the cestocidal activity of four South American Stevia species that previously showed antiprotozoal activity was analyzed using a motility assay on the laboratory cestode model, Mesocestoides vogae. The four Stevia extracts showed cestocidal activity, with S. alpina var. alpina as the most active. The sesquiterpene lactones estafietin and eupatoriopicrin were purified from S. alpina var. alpina and S. maimarensis, respectively, and tested on M. vogae. Estafietin showed cestocidal activity, inhibiting parasite viability in a dose-dependent manner, even from the first day of incubation. Consistent with the motility effects, the extract of S. alpina var. alpina and estafietin induced marked alterations in the morphology of the parasite. The results of this report show that Stevia species represent a source of new molecules with potential for the treatment of neglected tropical diseases caused by cestodes.

Bioethanol production from groundnut shell using microorganisms derived from sugarcane root soil

Bioethanol is a form of renewable energy that can be produced from fuel or energy crops. Ethanol is produced by the fermentation of sugars present in agricultural feed stocks and crop residues. This study investigates the use of agro wastes like groundnut shells for ethanol production. Initially, the groundnut shells were washed, dried and grinded into powder. Then it was subjected to ethanol production using yeast. After 20 days of incubation the ethanol was estimated using potassium dichromate method. The maximum ethanol yield (1.55%) was obtained when 1% yeast was used. To increase the efficiency of ethanol production, the cellulolytic bacteria was isolated from the cow dung dumped soil site. The 10 bacterial isolates were screened for cellulase enzyme production. Among them one bacterium showed maximum decolourization of congored which was subjected for enzyme production in nutrient broth. The organism showed maximum enzyme activity of 558.12 U/ml. The isolated cellulolytic bacteria were identified as Bacillus anthracis using 16s rDNA sequencing. Ethanol production from the groundnut shells was again carried out with various concentration of crude cellulase enzyme isolated from the bacteria. The estimation result showed 3.8% of ethanol as maximum. Then the ethanol was condensed using a rotary evaporator and when again estimated, showed 7.3% of ethanol. Finally, the presence of ethanol was confirmed by iodoform test. Thus, groundnut shells can efficiently be used for the production of ethanol, which can be used as a high potential fuel source for transportation in the future.

Use of Cannabis sativa L. for Improving Cadmium-Contaminated Mediterranean Soils—Effect of Mycorrhizal Colonization on Phytoremediation Capacity

Although the phytoremediation strategy has been studied worldwide, little research data are available regarding the influence of mycorrhizae on the phytoremediation capacity of various plants grown in Cd-contaminated soils in Mediterranean environments. Therefore, a pot experiment was carried out to study the possible effectiveness of hemp plant (Cannabis sativa L.) in the remediation of moderately and heavily Cd-contaminated soils and additionally to quantify the effect of Cd on Arbuscular Mycorrhizal Fungi (AMFs). For this purpose, an alkaline clay soil collected from the Farm of Institute of Plant Breeding and Genetic Resources (North Greece) was contaminated with two levels of Cd (3 and 30 mg Cd kg−1, corresponding to Levels A and B, respectively—first factor) at two incubation times (10 and 30 days—second factor) and six treatments (Control_30d, Control_10d, CdA_30d, CdB_30d, CdA_10d, CdB_10d) were created. Soil Cd concentrations, both pseudo-total and available to plants, were determined after extraction with Aqua Regia mixture and DTPA solution, respectively, before and after the cultivation of hemp plants and after the harvesting. Cd concentrations in the aboveground and underground plant parts were also estimated after digestion with Aqua Regia, while root colonization by AMFs was determined with a microscope. The highest plant’s Cd concentration, more than 50%, was observed in its underground part, at all Cd-contaminated treatments, indicating a strong capacity for cadmium to gather up in the roots. Among different Cd levels and incubation days, significant differences were recorded in the rates of root colonization by AMFs. Among different Cd levels and incubation days, 3 mg Cd Kg−1 soil promoted AMF root colonization, particularly at 10-day incubation, while 30 mg Cd Kg−1 soil diminished it. Colonization was lower with longer incubation times at both levels of Cd. Hemp appears to be a viable option for phytostabilization in Cd-contaminated soils, enabling further utilization of AMFs to assist the phytoremediation process.

إنتاج الليبازالبارد – النشط بواسطة فيوزاريوم سولاني

The current study aimed to the production and partial purification of a cold-active lipase by some fungi isolated from the olive oil processing wastes in Al-Gabal Al-Gharby, Libya. 31 fungal species from 12 genera were isolated. F.solani was the most prevalent comprising 94% of total Fusarium and 28.7% of total fungi, 102 fungal isolates were tested for their lipolytic activity on lipase production agar medium at 10 and 20°C. The most active isolates were Alternaria (2 isolates), Fusarium, and Penicillium (1isolate for each one). Molecular identification of the most active four isolates was carried out by their sequencing (ITS). The four powerful fungal strains' production of cold-active lipase was maximized by optimizing some nutritional and environmental factors. F. solani AUMC 16063 was able to produce the maximum amount of lipase activity (46.66U/mL/min) with specific activity (202.8U/mg), utilizing ammonium sulphate as a nitrogen source after 8 days of incubation at pH 3.0 and 15°C. However, at same condition after 6 days when yeast extract was employed as a nitrogen source, the generated cold-active lipase displayed the highest specific activity of (1550U/mg) and lipase activity (36.74U/ml/min). This is the first study in which the production, partial purification, maximized and characterization of a cold-active lipase enzyme by Fusarium solani.

Fungal secondary metabolite gliotoxin enhances enzymatic activity in soils by reshaping their microbiome

Gliotoxin (GT) is a sulfur-containing epidithiodioxopiperazine produced by various filamentous fungi, including those used in biological plant protection (Trichoderma virens). The pronounced antimicrobial effect of GT on a variety of fungi and bacteria makes it a promising agent for controlling phytopathogens in agricultural systems. In this study, we aim to investigate the microbiological properties of the soil microbiome after the introduction of GT. GT was applied at doses of 10, 25, 50, 100 and 500 μM kg−1 soil. Soil sampling was carried out after 1, 7, 14, 30, 60 and 90 days of incubation. It was found that GT significantly stimulated the respiratory activity of soil microorganisms and maintained this activity throughout the experiment. Carbon of microbial biomass, on the contrary, decreases under the influence of GT and is restored at the end of the experiment only in microcosms with 10 and 25 μM GT. Separate estimates of bacterial and fungal biomass showed that the bacterial community increased in biomass on day 14, while fungal biomass increased on day 30 after the treatment. Under the influence of GT, the activity of soil enzymes involved in the carbon (CB, βG, βX), nitrogen (NAG, LAP) and phosphate (AP) cycles significantly increased. High-throughput amplicon sequencing of the ITS and 16S rDNA markers revealed that the soil fungal community is more susceptible to GT than the bacterial community. This was reflected in changes in alpha-diversity indices and in the pattern of changes in the abundance of some microbial genera. Thus, on the one hand, the data obtained provides insight into the biological effects of GT on the soil microbial community. On the other hand, it sets the direction for further research into the ecological role of antibiotics produced by soil and rhizosphere microorganisms.

Half substitution of mineral N with fish protein hydrolysate enhancing microbial residue C and N storage and climate benefits under high straw residue return

The widespread utilization of straw return was a popular practice straw disposal for highly intensive agriculture in China, which has brought about some negative impacts such as less time for straw complete biodegradation, aggravation of greenhouse gas evolution, and lower efficient of carbon accumulation. It was urgent to find an eco-friendly N-rich organic fertilizer instead of mineral N as activator to solve the above problems and lead a carbon accumulation in long tern management. Besides, microbial necromass was considered as a crucial contributor to persistent soil carbon (C) and nitrogen (N) pool. How organic fertilizer activators influence microbial residue under different amount of crop residues input remained unclear. Thus, soils incorporating moderate and high rate of rice straw residue with additions of half and full of organic activators (fish protein hydrolysates vs. manure) were incubated for measuring carbon dioxide (CO2) and nitrous oxide (N2O) emission, microbial community and necromass. It was found that soil CO2 emission was rapidest during the first 13 days of straw decomposition but remained lowest in the treatments of 50% mineral N substituted by fish protein hydrolysate. There were that 81%–89% of total CO2 release and 59%–65% of total N2O emission occurred within 60 days of incubation period, and bacterial community and nitrate positively affected soil CO2 and N2O release respectively. Straw incorporation amount and organic activator application interactively influenced soil CO2 emission but not affected soil N2O emission. After 360 days of incubation, the difference of bacterial necromass was noticeable but fungal necromass remained almost unaltered across all treatments. All treatments showed generally comparable contribution of microbial necromass N to the total N pool. The treatment of 50% mineral N substituted by fish protein hydrolysate under high rate of straw input (HSF50) promoted the highest proportion of microbial necromass C in soil organic C because of alleviating N limitation for microorganisms. Finally, HSF50 was recommended as an eco-friendly strategy for enhancing microbial necromass C and N storage and climate benefits in agroecosystems.

Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates from a longitudinal study

Antibiotic tolerance in Mycobacterium tuberculosis reduces bacterial killing, worsens treatment outcomes, and contributes to resistance. We studied rifampicin tolerance in isolates with or without isoniazid resistance (IR). Using a minimum duration of killing assay, we measured rifampicin survival in isoniazid-susceptible (IS, n=119) and resistant (IR, n=84) isolates, correlating tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs), and isoniazid-resistant mutations. Longitudinal IR isolates were analyzed for changes in rifampicin tolerance and genetic variant emergence. The median time for rifampicin to reduce the bacterial population by 90% (MDK90) increased from 1.23 days (IS) and 1.31 days (IR) to 2.55 days (IS) and 1.98 days (IR) over 15–60 days of incubation, indicating fast and slow-growing tolerant sub-populations. A 6 log10-fold survival fraction classified tolerance as low, medium, or high, showing that IR is linked to increased tolerance and faster growth (OR = 2.68 for low vs. medium, OR = 4.42 for low vs. high, p-trend = 0.0003). High tolerance in IR isolates was associated with rifampicin treatment in patients and genetic microvariants. These findings suggest that IR tuberculosis should be assessed for high rifampicin tolerance to optimize treatment and prevent the development of multi-drug-resistant tuberculosis.

Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates from a longitudinal study.

Antibiotic tolerance in Mycobacterium tuberculosis reduces bacterial killing, worsens treatment outcomes, and contributes to resistance. We studied rifampicin tolerance in isolates with or without isoniazid resistance (IR). Using a minimum duration of killing assay, we measured rifampicin survival in isoniazid-susceptible (IS, n=119) and resistant (IR, n=84) isolates, correlating tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs), and isoniazid-resistant mutations. Longitudinal IR isolates were analyzed for changes in rifampicin tolerance and genetic variant emergence. The median time for rifampicin to reduce the bacterial population by 90% (MDK90) increased from 1.23 days (IS) and 1.31 days (IR) to 2.55 days (IS) and 1.98 days (IR) over 15-60 days of incubation, indicating fast and slow-growing tolerant sub-populations. A 6 log10-fold survival fraction classified tolerance as low, medium, or high, showing that IR is linked to increased tolerance and faster growth (OR = 2.68 for low vs. medium, OR = 4.42 for low vs. high, p-trend = 0.0003). High tolerance in IR isolates was associated with rifampicin treatment in patients and genetic microvariants. These findings suggest that IR tuberculosis should be assessed for high rifampicin tolerance to optimize treatment and prevent the development of multi-drug-resistant tuberculosis.

Embryonic Leucine Promotes Early Postnatal Growth via mTOR Signalling in Japanese Quails.

Nutritional cues during embryonic development can alter developmental trajectories and affect postnatal growth. However, the specific mechanisms by which nutrients influence avian growth remain largely unknown. Amino acids can directly interact with the nutrient-sensing pathways, such as the insulin-like growth factor 1 (IGF-1)/mechanistic target of rapamycin (mTOR) pathways, which are known to regulate growth. We examined the effects of embryonic leucine on gene expression and phenotypic growth in Japanese quails by injecting 2.5 mg leucine or saline (control) into Japanese quail eggs on the tenth day of incubation and incubating them under standard conditions. The treatment groups had similar hatching success and size at hatching. However, between 3 and 7 days post-hatching, quails treated with embryonic leucine showed increased growth in body mass and wing, tarsus, head, and intestinal lengths, lasting up to 21 days. The hepatic expression of IGF1, IGF1R, mTOR, and RPS6K1 was upregulated in leucine-treated quails, while the expression of FOXO1 remained unaffected. In conclusion, a subtle increase in embryonic leucine may induce developmental programming effects in Japanese quail by interacting with the IGF-1/mTOR nutrient-sensing pathway to promote growth. This study highlights the role of embryonic amino acids as crucial nutrients for enhancing growth. It provides valuable insight into nutrient intervention strategies during embryonic development to potentially improve poultry growth performance.

A 3D micro-computed tomography study comparing embryonic skeletal development in layer versus broiler strains of the domestic chicken

Our objective was to analyze the effect of selection for divergent traits in the domestic chicken on embryonic skeletal development, which could affect postnatal bird welfare. Development was compared between the Ross 308 broiler line (fast growth and muscle mass accrual) and Novoponte layers (high laying rate and egg quality). In Study 1 (Initial Conditions), we characterized egg composition prior to incubation and identified the onset of embryonic skeletal mineralization in the 2 strains. In Study 2 (Developmental Dynamics), we used 3D X-ray tomographic imaging (µCT) on incubation days ED11, ED13, ED15 and ED17 to track skeletal maturation trajectories as a pseudo-time series. Results showed that Ross 308 embryos, which are heavier than Novoponte embryos, possess higher levels of yolk nutrients including phosphorus and calcium, but lower eggshell mineral content, than Novoponte embryos. Skeletal mineralization started synchronously in both strains, on ED11. The higher mineral ion content in the larger yolk of Ross 308 eggs compared to Novoponte eggs may partly explain why skeletal mineralization in Ross 308 embryos advances faster: using µCT, we show that the mandible and tibiotarsi in Ross 308 embryos are larger at ED11 and ED13 compared with Novoponte embryos. However, Novoponte embryos catch up from this initial lag in mineralization by ED15. The timing of the Novoponte acceleration coincides with the functional activation of the chorioallantoic membrane in releasing calcium from the inner eggshell. This correlates with a decrease in eggshell thickness from ED11 to ED17 in Novoponte eggs, which was not observed during Ross 308 incubation. To conclude, while some temporal discrepancies exist in early skeletal development between the embryos of Ross 308 and Novoponte strains, overall prenatal skeletal maturation seems to be robustly regulated. Despite selection for antagonist production traits, layer and broiler prehatch skeletal morphology ultimately synchronizes. Practically, since the extent of skeletal maturity equalizes between strains towards the end of incubation, refinements of farming practices, postnatal environment, and diet should be considered for improving domestic fowl welfare.

Improved surrogate eggshell incubation system for assisted reproductive technologies such as cloning, genome modification, and de-extinction efforts

A germinal disc located on the egg yolk surface drives genetic modification. Windowed and surrogate eggshell incubation methods have been developed, but these exhibit limited abilities to generate transgenic chickens. In the present study, we investigated the frequency of observing the germinal disc according to the preincubation positioning direction and time and found that it depended on those conditions, but only a few chicks (2.8–5.6%) hatched using the windowed method. Then, we attempted to improve surrogate method via one- or two-step procedures. All eggs, including surrogates that were 10 g heavier than the donor eggs, were obtained from a poultry flock of the same age. With the one-step surrogate method, where the donor egg was transferred only once through a 3.5 cm hole on the point end, at the first day of preincubation, into the surrogate egg, the survival rate at day 4 was 30.8%, and the hatching rate was 11.8%. With the two-step surrogate method (transfer was on the 1st and 4th day of incubation), the survival rate at day 4 was improved to 90.7%, and a hatching rate of 70.0% was achieved. Therefore, this method can be effective for in ovo artificial incubation.

The Assessment of Methyl Methanesulfonate Absorption by Amphipods from the Environment Using Lux-Biosensors.

The ability of aquatic mesofauna representatives involved in trophic chains to sorb and accumulate toxicants is important for understanding the functioning of aquatic ecosystems and for fishing industry. This study investigated the capacity of marine amphipod Gammarus oceanicus and freshwater amphipods Eulimnogammarus vittatus and Gammarus lacustris to absorb the DNA-alkylating agent methyl methanesulfonate (MMS). The presence of alkylating agents in the environment and in the tissues of the amphipods was determined using whole-cell lux-biosensor Escherichia coli MG1655 pAlkA-lux, in which the luxCDABE genes from Photorhabdus luminescens, enabling the luminescence of the cell culture, are controlled by the PalkA promoter of DNA glycosylase. It was shown that within one day of incubation in water containing MMS at a concentration above 10 μM, the amphipods absorbed the toxicant and their tissues produce more alkylation damage to biosensor cells than the surrounding water. Concentrations of MMS above 1 mM in the environment caused the death of the amphipods before the toxicant could be significantly concentrated in their tissues. The sensitivity and the capacity to absorb MMS were found to be approximately the same for the marine amphipod G. oceanicus and the freshwater amphipods E. vittatus and G. lacustris.

MONITORING OF THE LIVING ENVIRONMENT THROUGH AIR POLLUTANTS IN THE POLOG FIELD OF THE REPUBLIC OF NORTH MACEDONIA

Introduction: The Republic of North Macedonia and especially the field pit of Polog in terms of geographical position and climatic aspect is considered as a place with different variables such as: temperature, humidity, radioactive exposure, hilly-mountainous relief. The Sharr mountain in the west and the dry mountain in the north, limit the Polog field pit in all directions, such as north-west and south-east, causing the living environment to deteriorate in the winter period due to air, water and land pollutants. With special emphasis, we highlight the winter season when we have an alarming state of air pollution, such as certain regions of the Republic of North Macedonia and especially the region of the field pit of Polog with the cities of Tetovo and Gostivar and the surrounding area. Recently, the city of Tetova and its surroundings is considered one of the most polluted cities in the country, region and the largest in the world, then this phenomenon of pollution necessarily leads us to unwanted consequences for the population that lives and operates in this region. As a result of environmental factors such as air pollution and other related factors such as: stress, temperature, food, social genes, mental state and other related factors can lead to disorders in the human body and lead to diseases that attack the nervous and cardiovascular systems , skeletal and thyroid. Many studies talk about the alarming condition of patients who are affected by these diseases, therefore our paper will focus on the effect of these factors above all on the actual state of pollutants in the Polog region of North Macedonia. The purpose of the study: This paper aims to investigate the potential causes that can affect the health of the population of the Polog field pit and especially the city of Tetova and its surroundings. As a potential pollutants of the living environment, the following can be considered: carbon monoxide, carbon dioxide, sulfur dioxide, urban combustion from automobiles, combustion of various physical substances, chemical components from factories, smoke from chimneys from burning oil and coal, combustion from wood in the form of pellets, etc. Material and working method: In the study, fertilized chicken eggs were taken and incubated in the incubator for 21 days of incubation. The eggs were treated with different doses of air pollutant extract at certain stages of embryonic development. Using the stereomicroscope with macroscopic and microscopic method were analyzed ontogenetic changes in certain stages of embryonic development. Research results: The results of the research will give a real insight into the situation of the level of pollution of the living environment in the city of Tetovo and its surroundings and in the field pit of Pollog. The monitoring of the living environment can be followed by the treatment of air pollutants in certain doses in the form of ampoule therapy, and the negative effect of the given dose on the hen's eggs will be observed. All eggs with the highest dose of injection had a stagnated embryonic development, whereas egss with lower dose of injections had underdeveloped embryos. Conclusion: From the results of the research we can conclude that the potential pollutants from air pollutants can lead to consequences for the population living in this region but also for the living world in general. These factors can lead to disorders of the nervous system, cardiovascular, and thyroid disorders and other respiratory diseases. This research will give a real insight into the pollution of the living environment and the possible consequences of this at a local, regional and wider level.