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Veterinary World Jan 2024The production of lignocellulosic biomass waste in the agricultural sector of Indonesia is quite high annually. Utilization of lignocellulosic biomass waste through...
BACKGROUND AND AIM
The production of lignocellulosic biomass waste in the agricultural sector of Indonesia is quite high annually. Utilization of lignocellulosic biomass waste through fermentation technology can be used as feed and biofuel. Fermentation technology requires the involvement of micro-organisms such as bacteria (lactic acid bacteria or LAB). LABs can be isolated from various sources, such as duck excreta. However, there have not been many reports of LAB from duck excreta. The present study aimed to characterize LAB enzymes isolated from duck excreta and obtain LAB enzymes with superior fermentation properties.
MATERIALS AND METHODS
A total of 11 LAB cultures obtained from duck excreta in Yogyakarta, Indonesia, were tested. Enzyme characterization of each LAB was performed using the API ZYM kit (BioMérieux, Marcy-I'Etoile, France). The bacterial cell suspension was dropped onto the API ZYM™ cupule using a pipette and incubated for 4 h at 37°C. After incubation, ZYM A and ZYM B were dripped onto the API ZYM cupule, and color changes were observed for approximately 10 s under a strong light source.
RESULTS
Esterase activity was moderate for all LABs. The activity of α-chymotrypsin, β-glucuronidase, α-fucosidase, and α-mannosidase was not observed in a total of 10 LAB. The phosphohydrolase and amino peptidase enzyme activity of seven LABs was strong. Only six LAB samples showed protease activity. The glycosyl hydrolase (GH) activity was observed in a total of 8 LAB, while the activity of 2 LAB was strong ( subsp. K5 and M4A).
CONCLUSION
A total of 2 LABs have superior properties. subsp. K5 and M4A have a high potential to be used in fermentation. They have the potential for further research, such as their effectiveness in fermentation, lignocellulose hydrolysis, feed additives, molecular characterization to detect specific enzymes, and their specific activities.
PubMed: 38406367
DOI: 10.14202/vetworld.2024.143-149 -
Biotechnology Journal Feb 2024Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more...
Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more accurately. The formation of 3D cell spheroids using hanging drops has emerged as a cost-effective and crucial method for generating uniformly-sized spheroids. This study aimed to validate the potential of a tip-refill wafer (TrW), a disposable laboratory item used to hold pipette tips, in facilitating 3D cell culture. The TrW allows for easy generation of hanging drops by pipetting the solution into the holes of the wafer. The mechanical stability of the hanging drops is ensured by the surface wettability and thickness of the TrW. Hanging drops containing 60-µL of solution remained securely attached to the TrW even when subjected to orbital shaking at 210 rpm. The exceptional resistance to mechanical shaking enabled the use of inertial focusing to facilitate spheroid formation. This was demonstrated through live/dead cell staining, quantitative polymerase chain reaction (qPCR) analysis, and cytoskeleton staining, which revealed that horizontal orbiting at 60 rpm for 15 min promoted cell aggregation and ultimately led to the formation of 3D spheroids. The spheroid harvest rate is 96.1% ± 3.5% across three TrWs, each containing 60 hanging drops. In addition to generating mono-culture 3D spheroids, the TrW-based hanging drop platform also enables the formation of multicellular spheroids, and on-demand pairing and fusion of spheroids. The TrW is a disposable item that does not require any fabrication or surface modification procedures, further enhancing its application potential in conventional biological laboratories.
Topics: Spheroids, Cellular; Cell Culture Techniques
PubMed: 38403449
DOI: 10.1002/biot.202300427 -
Biotechnology Journal Feb 2024Here, we developed a field-deployable molecular diagnostic kit for the detection of RNA viruses that operates in a pipette-free manner. The kit is composed of acrylic...
Here, we developed a field-deployable molecular diagnostic kit for the detection of RNA viruses that operates in a pipette-free manner. The kit is composed of acrylic sticks, PCR tubes, and palm-sized three-dimensional(3D)-printed heaters operated by batteries. The kit performs RNA extraction, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), and visual detection in one kit. An acrylic stick was engraved with one shallow and one deep cylindrical chamber at each end for the insertion of an FTA card and ethidium homodimer-1 (EthD-1), respectively, to perform RNA extraction/purification and bimodal visual detection of the target amplicons. First, an intercalation of EthD-1 into the target DNA initially produces fluorescence upon UV illumination. Next, the addition of a strong oxidant, in this case sodium (meta) periodate (NaIO ), produces intense aggregates in the presence of EthD-1-intercalated DNA, realized by electrostatic interaction. In the absence of the target amplicon, no fluorescence or aggregates are observed. Using this kit, two major infectious viruses-severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus (SARS-CoV-2)-were successfully detected in 1 h, and the limits of detection (LOD) were approximately 1 virus μL for SFTSV and 10 copies μL for SARS-CoV-2 RNA. The introduced kit is portable, end-user-friendly, and can be operated in a pipette-free manner, paving the way for simple and convenient virus detection in resource-limited settings.
Topics: Humans; RNA, Viral; Pathology, Molecular; Sensitivity and Specificity; COVID-19; Virus Diseases; Nucleic Acid Amplification Techniques; DNA; COVID-19 Testing
PubMed: 38403439
DOI: 10.1002/biot.202300521 -
Proceedings. Biological Sciences Feb 2024Sabotaging milkweed by monarch caterpillars () is a famous textbook example of disarming plant defence. By severing leaf veins, monarchs are thought to prevent the flow...
Sabotaging milkweed by monarch caterpillars () is a famous textbook example of disarming plant defence. By severing leaf veins, monarchs are thought to prevent the flow of toxic latex to their feeding site. Here, we show that sabotaging by monarch caterpillars is not only an avoidance strategy. While young caterpillars appear to avoid latex, late-instar caterpillars actively ingest exuding latex, presumably to increase sequestration of cardenolides used for defence against predators. Comparisons with caterpillars of the related but non-sequestering common crow butterfly () revealed three lines of evidence supporting our hypothesis. First, monarch caterpillars sabotage inconsistently and therefore the behaviour is not obligatory to feed on milkweed, whereas sabotaging precedes each feeding event in caterpillars. Second, monarch caterpillars shift their behaviour from latex avoidance in younger to eager drinking in later stages, whereas caterpillars consistently avoid latex and spit it out during sabotaging. Third, monarchs reared on detached leaves without latex sequestered more cardenolides when caterpillars imbibed latex offered with a pipette. Thus, we conclude that monarch caterpillars have transformed the ancestral 'sabotage to avoid' strategy into a 'sabotage to consume' strategy, implying a novel behavioural adaptation to increase sequestration of cardenolides for defence.
Topics: Animals; Larva; Latex; Butterflies; Asclepias; Cardenolides
PubMed: 38378155
DOI: 10.1098/rspb.2023.2721 -
BMC Pediatrics Feb 2024Neonatal jaundice is a condition caused by elevated levels of bilirubin in the bloodstream. Laboratory determination of serum bilirubin concentration by total serum... (Observational Study)
Observational Study
BACKGROUND
Neonatal jaundice is a condition caused by elevated levels of bilirubin in the bloodstream. Laboratory determination of serum bilirubin concentration by total serum bilirubin (TSB) test is still considered as gold standard for clinical guidance and practice. In developed countries, diagnosis of neonatal jaundice is shifting towards point-of-care medical devices. BiliDx is a device developed to allow a fast, blood-based determination of bilirubin levels at the point of care. This study aimed to determine the accuracy of the BiliDx device relative to a standard laboratory total serum bilirubin to diagnose and monitor jaundice among neonates admitted at Muhimbili National Hospital (MNH).
MATERIAL AND METHODOLOGY
This was a prospective hospital-based observational study conducted at the Neonatal Ward - MNH, Dar-es-Salaam, Tanzania from November 2022 to January 2023. A total of 180 neonates admitted at the neonatal ward with jaundice and whose parents consented were enrolled in the study. Blood samples were collected; 2 ml of venous blood into the vacutainer bottle for standard laboratory measurement of total serum bilirubin (TSB) and 25µL blood collected into a transfer pipette tube and applied to BiliDx. STATA version 15.1 was used for data analysis.
RESULTS
Out of 180 neonates, 39.4% (71/180) had birth weight between 1500 - 2499.9 g, approximately 2/3rd (120/180) were preterm, 92/180 (51.1%) were males and 100/180 (55.6%) were undergoing phototherapy treatment the moment sample taken. The mean bilirubin concentration was 92 mmol/l for BiliDx and 118 mmol/l for standard laboratory TSB. The minimum and maximum values obtained with BiliDx were, 3.4 and 427.5 mmol/l respectively, compared with 10.7 and 382.1 mmol/l using standard laboratory TSB. A linear relationship and correlation coefficient of 0.8408 (p = 0.000) between BiliDx and standard laboratory TSB was found. The regression analysis showed the presence of constant error [coefficient of BiliDx/slope = 0.91, 95% CI (0.82-0.99), p = 0.000] and random error exclusively [coefficient of constant/y-intercept = 48.52, 95%CI (37.70-59.34), p = 0.000]. The Bland-Altman plot showed an acceptable mean difference of 39.1mmol/l, limits of agreement of -48.3mmol/l to 126.4mmol/l, and 179 points (179/180 = 99.4%) lying inside the limits of agreement.
CONCLUSION
The results support the use of BiliDx for rapid and accurate testing of elevated levels of bilirubin in the bloodstream among neonates since 99.4% of the differences between BiliDx and standard laboratory TSB lie between the lines of agreement.
Topics: Infant, Newborn; Male; Humans; Female; Jaundice, Neonatal; Bilirubin; Point-of-Care Systems; Prospective Studies; Jaundice; Phototherapy; Hospitals; Neonatal Screening
PubMed: 38350890
DOI: 10.1186/s12887-024-04565-w -
Diagnostic Microbiology and Infectious... Apr 2024We hypothesized that the loop material and size could affect the results of the culture when compared to the calibrated pipette. A total of 484 urine samples were...
We hypothesized that the loop material and size could affect the results of the culture when compared to the calibrated pipette. A total of 484 urine samples were included in the study, and each sample was plated by using different loop types and the calibrated pipette. The bacterial counts per milliliter were calculated and compared, with a focus on the important cutoff values of 10³ and 10⁴ CFU/ml for further identification. When considering the 10³ CFU/ml as cutoff value, 1 µl and 10 µl plastic loops gave the highest sensitivity (86.8 %), whereas the 10 µl metal loop had the lowest sensitivity (64.2 %). For the 10⁴ CFU/ml cutoff value, 1 µl plastic loop inoculation demonstrated the highest sensitivity (75.9 %), while the 10 µl metal loop provided the lowest sensitivity (26.5 %). These results suggest that the single use plastic loops are functional, sensitive, useful especially for critical sample.
Topics: Humans; Urinary Tract Infections; Urinalysis; Bacterial Load; Urine Specimen Collection; Urine; Sensitivity and Specificity
PubMed: 38330685
DOI: 10.1016/j.diagmicrobio.2024.116192 -
Cytoskeleton (Hoboken, N.J.) Feb 2024Although diverse actin network architectures found inside the cell have been individually reconstituted outside of the cell, how different types of actin architectures...
Although diverse actin network architectures found inside the cell have been individually reconstituted outside of the cell, how different types of actin architectures reorganize under applied forces is not entirely understood. Recently, bottom-up reconstitution has enabled studies where dynamic and phenotypic characteristics of various actin networks can be recreated in an isolated cell-like environment. Here, by creating a giant unilamellar vesicle (GUV)-based cell model encapsulating actin networks, we investigate how actin networks rearrange in response to localized stresses applied by micropipette aspiration. We reconstitute actin bundles and branched bundles in GUVs separately and mechanically perturb them. Interestingly, we find that, when aspirated, protrusive actin bundles that are otherwise randomly oriented in the GUV lumen collapse and align along the axis of the micropipette. However, when branched bundles are aspirated, the network remains intact and outside of the pipette while the GUV membrane is aspirated into the micropipette. These results reveal distinct responses in the rearrangement of actin networks in a network architecture-dependent manner when subjected to physical forces.
PubMed: 38326972
DOI: 10.1002/cm.21836 -
Theriogenology Apr 2024The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques....
The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques. While trophectoderm (TE) biopsies are frequently used for genotyping, cell-free DNA (cfDNA) found in blastocoele fluid (BF) arises as a less-invasive method. Moreover, the blastocoel collapse produced by BF aspiration could be beneficial for embryo cryotolerance. This study was conducted to test the BF as a source of cell free-DNA (cfDNA) and to compare the BF to the TE biopsy in terms of sexing efficiency/accuracy, embryo survival and gene expression after vitrification/warming. IVP day 7 expanded blastocysts were artificially collapsed by aspiration of BF (VIT-Collapsed) or biopsied (VIT-Biopsied). After sample collection, embryos were vitrified/warmed by the Cryotop method and individually cultured in vitro. Intact fresh non-vitrified and vitrified/warmed blastocysts served as Fresh Control and VIT-Control, respectively. After sex identification of BF or TE biopsies and the corresponding surviving embryos, amplification efficiency and sexing accuracy were assessed. There were no differences between the BF and TE biopsy samples in terms of sexing accuracy or efficiency. Although all vitrified groups showed lower post-warming re-expansion rates (p < 0.05), the blastocyst re-expansion rates in the VIT-Collapsed group were comparable to those in the Fresh Control group whereas biopsied blastocysts showed the lowest (p < 0.05) re-expansion rates. VIT-Collapsed blastocysts had hatching rates that were comparable to those of Fresh Control blastocysts but significantly higher than those of the other vitrification treatments. Proapoptotic gene BAX was overexpressed in VIT-Biopsied embryos, whereas BCL2 transcripts were more abundant in the VIT-Collapsed group. On the other hand, VIT-Biopsied embryos showed altered ATP1B1- and AQP3-mRNA levels. The analysis of the cfDNA present in the BF is an efficient, minimally invasive approach to sex IVP cattle embryos. Besides, the artificial collapse of blastocoel prior to vitrification resulted in higher re-expansion and hatching ability than when embryos were vitrified after being biopsied.
Topics: Cattle; Animals; Vitrification; Cryopreservation; Blastocyst; Biopsy; Cell-Free Nucleic Acids
PubMed: 38325151
DOI: 10.1016/j.theriogenology.2024.01.042 -
Asian Journal of Andrology Feb 2024Ex vivo tissue culture of the human corpus cavernosum (CC) can be used to explore the tissue structural changes and complex signaling networks. At present, artificial...
Ex vivo tissue culture of the human corpus cavernosum (CC) can be used to explore the tissue structural changes and complex signaling networks. At present, artificial CC-like tissues based on acellular or three-dimensional (3D)-printed scaffolds are used to solve the scarcity of primary penis tissue samples. However, inconvenience and high costs limit the wide application of such methods. Here, we describe a simple, fast, and economical method of constructing artificial CC-like tissue. Human CC fibroblasts (FBs), endothelial cells (ECs), and smooth muscle cells (SMCs) were expanded in vitro and mixed with Matrigel in specific proportions. A large number of bubbles were formed in the mixture by vortexing combined with pipette blowing, creating a porous, spongy, and spatial structure. The CC FBs produced a variety of signaling factors, showed multidirectional differentiation potential, and grew in a 3D grid in Matrigel, which is necessary for CC-like tissue to maintain a porous structure as a cell scaffold. Within the CC-like tissue, ECs covered the surface of the lumen, and SMCs were located inside the trabeculae, similar to the structure of the primary CC. Various cell components remained stable for 3 days in vitro, but the EC content decreased on the 7th day. Wingless/integrated (WNT) signaling activation led to lumen atrophy and increased tissue fibrosis in CC-like tissue, inducing the same changes in characteristics as in the primary CC. This study describes a preparation method for human artificial CC-like tissue that may provide an improved experimental platform for exploring the function and structure of the CC and conducting drug screening for erectile dysfunction therapy.
PubMed: 38319194
DOI: 10.4103/aja202374 -
Cryo Letters 2023Amides are low molecular weight cryoprotectants. N-methylacetamide (MA) is one of the cryoprotectant agents in this group.
BACKGROUND
Amides are low molecular weight cryoprotectants. N-methylacetamide (MA) is one of the cryoprotectant agents in this group.
OBJECTIVE
To investigate the cryoprotective effect of MA in rabbit semen.
MATERIALS AND METHODS
For this purpose, six ejaculates from six New Zealand rabbits were collected and pooled using an artificial vagina. Pooled semen was divided into four equal parts and diluted with TCG+ egg yolk. CPA was added to form the following groups: Control with 6% DMSO; Group 1 with 1% MA; Group 2 with 2% MA; and Group 3 with 3% MA. After the addition of CPA, the semen eqilibration procedure was started. Sperm were then drawn into 0.25 mL straws, frozen by automatic semen freezing and stored in a liquid nitrogen container. Pipettes were thawed after 24 h and analyses were performed.
RESULTS
Total, progressive and rapid motility values of the Control group were higher than those of the MA groups (p<0.05). However, there was no statistical difference between the Control and Group 2 in terms of these parameters. While there was no statistical difference between the groups in terms of acrosome damage and mitochondrial membrane potential, the best results were observed in Control, Group 2, Group 1 and Group 3, respectively. When we compared all groups, no difference was found in terms of MDA, CAT and GSH-Px. There was a statistical difference between Group 3 and the Control in terms of GSH level (p<0.05).
CONCLUSION
DMSO appeared to be more useful for the cryopreservation of rabbit semen compared to MA. Doi.org/10.54680/fr23610110812.
Topics: Female; Rabbits; Male; Animals; Cryoprotective Agents; Semen; Dimethyl Sulfoxide; Cryopreservation; Semen Preservation; Sperm Motility; Spermatozoa; Semen Analysis; Acetamides
PubMed: 38311932
DOI: No ID Found