Gel Electrophoresis Research Articles

Detection of anti-leptospiral antibodies using recombinant ErpY-like lipoprotein based latex agglutination test for serodiagnosis of animal leptospirosis.

Precise and timely diagnosis is essential to prevent severe outcomes of leptospirosis in humans and animals. Existing diagnostic methods face challenges and limitations, underscoring the need for novel, field-applicable screening, and diagnostic tests/assays. This study evaluates the diagnostic utility of a recombinant ErpY-like lipoprotein (rErpY-LIC11966) in a latex agglutination test (LAT) for diagnosis of animal leptospirosis. The ErpY gene sequence from Leptospira interrogans serovar Pomona, excluding the signal peptide, was amplified, cloned into the pETite vector, and expressed in Escherichia coli. The expressed rErpY (∼16kDa) was characterized by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western blot using Leptospira-specific standard sera. To assess the diagnostic potential of rErpY, Ni-NTA affinity-purified protein was used to sensitize latex-coated beads (0.8µm colour beads), which were then employed in the LAT for standardization and optimization with standard positive and negative sera. For evaluation, the rErpY-LAT was tested on serum samples from 177 suspected animal cases and compared to the microscopic agglutination test. It showed a relative diagnostic sensitivity of 90.6%, a specificity of 89.1%, and an overall accuracy of 90%. This study proposes rErpY-LAT as a field testing/screening diagnostic tool for preliminary serodiagnosis of leptospirosis, highlighting the potential of recombinant protein-based assays to address current diagnostic challenges.

Aedes aegypti feeding behavior during dengue outbreaks in two rural areas of Peru during the Yaku cyclone and El Niño phenomenon of 2023.

Motivation for the study. Dengue epidemics caused by A. aegypti occur during climatic events in tropical countries such as Peru; however, the feeding behavior of the mosquito usually goes unnoticed. Main findings. A. aegypti populations in Marcavelica and Querecotillo showed anthropophilic feeding behavior during cyclone Yaku and in the 2023 El Niño. However, populations with different feeding patterns are not ruled out. Implications. The PCR-RFLP technique of the blood cell cytochrome B gene could be implemented in vector control policies through an entomo-virological surveillance plan. To determine the feeding behavior of Aedes aegypti in dengue outbreaks in two rural areas of Peru during the Yaku cyclone and El Niño phenomenon of 2023. Eight blood samples (8 pools) were obtained from the abdomen of 80 Aedes aegypti specimens captured in the rural districts of Querecotillo and Marcavelica during the Yaku cyclone and El Niño dengue outbreaks. DNA was extracted from the analyzed samples, then a PCR was directed at the CytB gene as a genetic marker and the PCR products were enzymatically digested with the restrictases Hae III and Mwo I. The PCR-RFLP products were visualized by agarose gel electrophoresis at 4%. DNA was obtained from all samples and a 358 bp amplicon was obtained as a PCR product. Likewise, the only RFLP found in Hae III was from Homo sapiens sapiens (233 and 125 bp). RFLP was not found in Hae III of Gallus gallus and RFLP in Mwo I of Canis familiaris and Mus musculus. Aedes aegypti showed conserved anthropophilic feeding behavior in dengue outbreaks in rural areas during the Yaku cyclone and El Niño.

Molecular Patterns and Antimicrobial Resistance Characterization of Salmonella enterica Non-Typhoidal from Human, Food, and Environment Samples Isolated in Luanda, Angola

The aim of this study was to characterize the antimicrobial resistance phenotype and genotype of non-typhoidal Salmonella spp. isolated in Luanda, Angola. Between 2013 and 2015, human clinical samples, food, and environmental samples (n = 290) were collected at different regions of Luanda city and screened for the presence of Salmonella spp. Bacterial isolates were preliminarily identified using the API 20E Kit, and their identification was confirmed using PCR and serotyping. All Salmonella spp. isolates were tested by minimum inhibitory concentration against 19 antimicrobials. The isolates were also screened using PCR for the presence of resistance genes (blaOXA-1, blaSHV, blaTEM, sul1, sul2, sul3, qnrA, qnrB, qnrS, qnrC, qnrD, aac(6′)-Ib, dfrIa [targeting dfrA1, dfrA5, dfrA15, dfrA15b, dfrA16, dfrA16b] and dfrA12, cmlA, and floR) and typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Salmonella enterica non-typhoidal was detected in 21.3% of the clinical samples (n = 32/150), 11.1% of the food samples (n = 10/90), and 26% of the environmental samples (n = 13/50). Serotyping revealed that the monophasic variant of Salmonella Typhimurium (Salmonella enterica serovar 4,[5],12:i:-) was detected in 38.1% of the samples. Moreover, serovar Salmonella Enteritidis was the second most frequent. Only 7.3% of the isolates were resistant to at least one antimicrobial. Furthermore, isolates from different origins (clinical, environmental, and food) were associated with the same lineages, Salmonella Enteritidis ST11 and S. enterica ser. Typhimurium ST313. The detection of S. enterica serovar 4,[5],12:i:- in different settings reinforces the need for a One Health approach to control this zoonosis in Angola.

Protein kinase D1 mitigation against etoposide induced DNA damage in prostate cancer is associated with increased α-Catenin.

The E-cadherin, α- and β-Catenin interaction at the cell adherens junction plays a key role in cell adhesion; alteration in the expression and function of these genes are associated with disease progression in several solid tumors including prostate cancer. The membranous β-Catenin is dynamically linked to the cellular cytoskeleton through interaction with α-Catenin at amino acid positions threonine 120 (T120) to 151 of β-Catenin. Nuclear presence of α-Catenin modulates the sensitivity of cells to DNA damage. The objective of this study is to determine the role of α-Catenin and protein kinase D1 (PrKD1) in DNA damage response. Prostate cancer cells; LNCaP, LNCaP (Sh-PrKD1; silenced PrKD1), C4-2 and C4-2 PrKD1 were used for various sets of experiments to determine the role of DNA damage in PrKD1 overexpression and silencing cells. These cells were treated with compound-10 (100 nM) and Etoposide (30 µM), total cell lysates, cytosolic and nuclear fractions were prepared to observe various protein expressions. We performed single cell gel electrophoresis (COMET assay) to determine the etoposide induce DNA damage in C4-2 and C4-2 PrKD1 cells. The animal experiments were carried out to determine the tolerability of compound-10 by mice and generate preliminary data on efficacy of compound-10 in modulating the α-Catenin and PrKD1 expressions in inhibiting tumor progression. PrKD1, a novel serine threonine kinase, phosphorylates β-Catenin T120. In silico analysis, confirmed that T120 phosphorylation alters β- to α-Catenin binding. Forced expression of PrKD1 in prostate cancer cells increased β- and α-Catenin protein levels associated with reduced etoposide induced DNA damage. Downregulation of α-Catenin abrogates the PrKD1 mitigation of DNA damage. The in vitro results were corroborated in vivo using mouse prostate cancer patient derived xenograft model by inhibition of PrKD1 kinase activity with compound-10, a selective PrKD inhibitor, demonstrating decreased total β- and α-Catenin protein levels, and β-Catenin T120 phosphorylation. Alteration in DNA damage response pathways play major role in prostate cancer progression. The study identifies a novel mechanism of α-Catenin dependent DNA damage mitigation role for PrKD1 in prostate cancer.

Surface Functionalization of Gold Nanoparticles Using Alkyne Derivatives: Applications in Chemical Sensing.

Colloidal gold nanoparticles (AuNPs) are important nanomaterials for chemical sensing and therapeutics. For their application, it is vital to develop a reliable and robust surface functionalization method that can be applied to diverse functional molecules and offer better stability under harsh biological conditions. Currently, thiol (SH) is the most commonly used functional group for forming stable covalent bonds with AuNPs. However, thiolated molecules typically require complicated preparation procedures, are susceptible to oxidation, and are not compatible with many electrophiles and reducing groups. In this study, we report that surface functionalization of AuNPs can be achieved using alkyne derivatives, which exhibit several advantages over classical thiolation and peptide-bond methods, including straightforward preparation of alkyne derivatives, rapid and simple conjugation in buffers and complex media, higher conjugation efficiency, long-term stability, and resistance to decomposition under harsh conditions. Several alkynylated biotin and fluorescein derivatives are prepared, and the alkynylated-AuNPs are characterized using a lateral flow assay, gel electrophoresis, and spectroscopy techniques to investigate the conjugation efficiencies, size distributions, protein interaction properties, and binding mode of the Au-alkyne bond. We also demonstrate that alkynylated-AuNPs can be used for the sensitive detection of hydrogen peroxide and streptavidin proteins.

Assessing the effects of NapA gene overexpression on denitrification and denitrogenation in magnetospirillum gryphiswaldense MSR-1.

Previous research on Magnetospirillum gryphiswaldense MSR-1 found that MSR-1 has a good denitrification nitrogen removal ability and specific application prospects in the sewage biological nitrogen removal field. Therefore, this study selected the essential denitrification gene NapA in MSR-1-wt for overexpression, and the overexpressed MSR-1-NapA was successfully constructed. Q-PCR amplification experiment and AGAR gel electrophoresis experiment proved that the relative transcription level of the NapA gene was increased by more than four times, and the denitrification ability of MSR-1-wt and MSR-1-NapA was further determined by enzyme activity experiment, denitrification experiment, and flow cytometry. The results showed that overexpression of the NapA gene increased nitrate reductase activity in MSR-1-NapA by more than four times. In the solution with a nitrate concentration of 118.33 ± 3.23mgN/L, the denitrification efficiency of MSR-1-NapA was superior to that of MSR-1-wt, significantly enhancing both the denitrification and nitrogen removal capacities of MSR-1. This indicates its greater potential for biological nitrogen removal in wastewater treatment.

Loop-Mediated Isothermal Amplification Assay Using Gold Nanoparticles for Detecting Treponema pallidum subspp. pallidum.

Venereal syphilis in humans is caused by Trepenoma pallidum subspp. pallidum. A study has shown that 30,302 individuals in Thailand had syphilis in 2020, with a male-to-female ratio of 1:0.8 and the highest incidence rate at ages between fifteen and twenty-four. This research aimed to develop a loop-mediated isothermal amplification assay using gold nanoparticles (LAMP-AuNPs). Analytical sensitivity, diagnostic specificity, accuracy, and predictive values for each technique are provided. The diagnosis sensitivities of polymerase chain reaction using agarose gel electrophoresis (PCR-AGE), loop-mediated isothermal amplification assay using agarose gel electrophoresis (LAMP-AGE), and LAMP-AuNPs were 116 ng/µL, 11.6 ng/µL, and 11.6 ng/µL, respectively. We evaluated the analytical specificity using PCR and a LAMP-based assay, and there was no cross-reactivity to Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy humans. After analyzing 400 serum samples of patients suspected of syphilis, the LAMP-AGE and LAMP-AuNPs assays displayed 100% diagnostic sensitivity scores, 91% diagnostic specificity scores, 95.5% accuracy rates, 100% positive predictive values (PPVs), and 91% negative predictive values (NPVs), the positive likelihood ratio (LR+) was 11.11, while the negative likelihood ratio (LR-) was 0. Conversely, for PCR assays displayed 100% diagnostic sensitivity scores, 94.5% diagnostic specificity scores, 97.25% accuracy rates, 100% PPVs, and 94.5% NPVs, LR+ was 18.18, and LR- was 0. The LAMP-AuNPs technique demonstrates rapidity, affordability, and convenience, rendering it well-suited for point-of-care applications in the diagnosis, prevention, and management of pathogenic infections.

Elucidation of the Possible Molecular Mechanism for the Anti-Apoptotic Effect of Terpene-Rich Extract of Amomum subulatum and Coriandrum sativum against Rodent Model of Cardiotoxicity

Background: Spices, including Amomum subulatum and Coriander sativum, are not only popular for improving food flavour but are also accepted worldwide for their health benefits in various disorders. Objective: This study aimed to elucidate the possible mechanism for the anti-apoptotic effect of terpene-rich extract of Amomum subulatum and Coriandrum sativum against the rodent model of cardiotoxicity. Methods: Effects of standardized terpenoids-rich aqueous methanolic extract of two commonly used spices, viz. Amomum subulatum Roxb. and Coriandrum sativum Linn., were investigated on doxorubicin-induced apoptotic changes and cardiotoxicity in Wistar rats with the aim of in-vestigating the mechanism. Prior to the in vivo experiment, the extracts were subjected to quan-titative estimation of possible bioactive markers of the terpenes by employing a newly devel-oped, optimized, and validated GC-MS method along with TLC profiling. Results: Cardiotoxicity was evident from elevated creatinine kinase (CK-MB), lactate dehydro-genase (LDH), and malondialdehyde (MDA) levels in the toxic control group after treatment with doxorubicin (2.5mg/kg i.p. given twice a week for three weeks). Caspase-3, Tumor necrosis factor-α (TNF-α), and Interleukin-6 (IL-6) were also induced in animals treated with doxorubi-cin. Treatment with Amomum subulatum and Coriandrum sativum at the doses of 100 and 200mg/kg exhibited significant (P<0.001) reversal of CK-MB, LDH, MDA, Caspase-3, TNF-α, and IL-6 levels. This protective effect was further supported by the results of DNA gel electro-phoresis and histopathological observations. Conclusion: This study supports the cardioprotective role of selected spices against doxorubicin-induced cardiotoxicity through the anti-apoptotic mechanism.

Marker Peptides for Indicating the Spoilage of Milk-Sample Preparation and Chemometric Approaches for Yielding Potential Peptides in a Raw Milk Model.

The diminishing of food waste is gaining increasing importance, especially in context with a growing population and a need for the sustainable use of food resources. A more precise determination of the best-before date can contribute to this general aim. As proteoforms can be regarded as indicators for ecophysiological influences, their suitability for determining the spoilage and, consequently, the shelf-life of food is suggested. Proteoforms reflect the spoilage of food more accurately. The aim of the present study was to develop an efficient proteomics workflow to determine the shelf-life of milk as a prominent target. In this case, raw milk was chosen as model, as it degrades much faster. The integration of different multivariate analysis techniques was used to analyze the spoilage of raw milk with regard to aspects of its proteome. As the feasibility of such an approach has already been demonstrated in previous studies, it is further necessary to enable a robust and reproducible workflow, primarily gaining appropriate numbers and amounts of peptides when the research question differs and other dairy products are evaluated. In the present study, two approaches for gaining peptides were considered: In addition to a direct hydrolysis of a protein-rich sample solution, in-gel hydrolysis is another common approach in proteomics. By separating the proteins in a traditional gel electrophoresis before hydrolysis, the change in the individual proteins and, consequently, potential peptides can be monitored more specifically during storage. However, the traditional approach offers not only possibilities but also limitations that must be considered. The study showed that it is beneficial to apply a combination of different application strategies, as they complement each other and can thus increase the information content of a sample or confirm a theory. Mass spectrometric features, which represent a chemical-structural change of all kinds of compounds during storage, were selected, and three of them were identified as peptides, originating from α-s1-casein.

Aurelia aurita jellyfish collagen: Recovery properties

Wound and burn healing is a complex physiological process that can be facilitated by medications based on marine collagen. In this regard, biomass of the Aurelia aurita jellyfish is a promising alternative source of medical collagen. As the global incidence of burns and wounds continues to grow, new healing methods have become a relevant area of medical science. This study featured acetic acid as a means of marine collagen extraction from A. aurita biomass. The physical and chemical properties of jellyfish collagen were determined gravimetrically and included such indicators as water solubility and water holding capacity. The molecular weight was defined by gel electrophoresis. The spectral studies relied on the method of UV spectroscopy. The regenerative experiments included such parameters as cytotoxicity, antioxidant properties, adhesion, and wound healing rate, as well as a quantitative PCR analysis. The optimal conditions for maximal collagen yield were as follows: 0.5 M acetic acid and 48 h extraction time. However, the collagen yield was very low (≤ 0.0185%). The high water holding capacity showed good prospects for A. aurita collagen to be used as hemostatic sponge. The acid-soluble collagen sample had a molecular weight of 100–115 kDa, which made it possible to classify it as type I. A. aurita jellyfish collagen revealed no cytotoxic properties; it had no effect on adhesion, migration, and proliferation of keratinocytes, neither did it affect the expression of cell differentiation markers. The wound healing model proved that the marine collagen had regenerative properties as it was able to increase the wound healing rate by 24.5%. Therefore, collagen extracted from the biomass of A. aurita jellyfish d emonstrated good p rospects for cosmetology and regenerative medicine.

Evaluation of coinfection between SARS-CoV-2 and dengue virus in São Luís/MA

The COVID-19 pandemic has become a global public health emergency, especially in subtropical regions endemic for arboviruses. During the acute phase of the disease, Dengue and COVID-19 present similar laboratory profiles and symptomatology, complicating clinical diagnosis. This study analyzed the occurrence of coinfections between dengue virus and SARS-CoV-2 in patients treated at a laboratory in São Luís, Maranhão. A total of 150 random serum samples from patients, collected between April and May 2020, tested positive for IgM/IgA antibodies against SARS-CoV-2. For the detection of dengue virus (DENV), reactions were standardized using specific primers for each serotype (1-4). Subsequently, cDNA amplification was performed by conventional PCR, and the amplicons were analyzed by agarose gel electrophoresis. Through this study, it was possible to identify one sample (0.6%) positive for the DENV-4 serotype. This study validates the possibility of coinfections in dengue-endemic areas.

A Rare Case of Homozygous Hemoglobin G-Philadelphia & Alpha Thalassemia

Abstract Introduction/Objective Hemoglobin (Hb) G-Philadelphia (GPh) is an alpha chain gene variant caused by a missense mutation resulting in a change from Glu to Asp at position 68. This change leads to alterations in the Hb structure and oxygen affinity. The mutation is inherited in an autosomal codominant pattern allowing for an asymptomatic carrier state. While the most common alpha chain variant, GPh heterozygosity is still infrequent, estimated at 1:5000 in African Americans. In African Americans, GPh is frequently linked to an alpha thalassemia deletion. On its own it is considered a benign variant that does not result in significant health issues. Homozygous HbGPh frequency is estimated at 1 in 25 million. Methods/Case Report A postpartum woman in her 4th decade of life was found to have a Hb of 10.8 and a mean corpuscular volume of 74. Initial work up included Hb electrophoresis that quantified HbA at 0%, Hb F at 0%, HbA2 at 0%, and 2 Hb variants representing 96.1% and 3.8% of the total globin. The major Hb variant migrated in the D or G position by alkaline and acid gel Hb electrophoresis. The minor variant was consistent with HbG2. Due to this abnormal Hb electrophoresis, a sample was sent out for a Hb evaluation reflexive cascade (HERC). HERC analysis by high-performance liquid chromatography and capillary gel electrophoresis found 99.9% of Hb consistent with the alpha chain variant HbG. Analysis of the alpha globin gene region revealed 2 copies of an alpha globin gene deletion that was interpreted as alpha-thal trait (-a/-a). This result is consistent with homozygous alpha-thal-2 and homozygous HbGPh (alpha^G/thal + alpha^G/thal). Results (if a Case Study enter NA) NA Conclusion Alpha^G/thal + alpha^G/thal presents a unique and complex challenge for diagnosis due to its rarity and because HbG2 is not always visualized by alkaline gel electrophoresis. The presence of a Hb G or D variant, a HbG2 variant, and the lack of both HbA and HbA2 are key electrophoretic findings. Clinically, alpha^G/thal + alpha^G/thal behaves essentially like a homozygous alpha-thal-2 trait with persistent microcytosis and erythrocytosis. Thus, co- inheritance of the alpha-thal trait will be suggested by a thalassemic complete blood count pattern.

Effect of Ultrahigh Dose Rate on Biomolecular Radiation Damage.

Dose rate is one of the important parameters in radiation-induced biomolecular damage. The effects of dose rate have been known to modify radiation toxicity in biological systems. The rate and extent of sublethal DNA damage (e.g., base damage and single-strand breaks) repair and those of cell proliferation have been manifested by dose rate. However, the recent preclinical application of ultrahigh dose rate [(UHDR) ca. 40 Gy/s and higher] radiation modalities have been shown to lower the type and extent of radiation damage to biological systems. At these UHDR, radiation-induced physicochemical and chemical processes are expected to differ from those observed after irradiation at conventional dose rates (CONV). It is unclear whether these UHDR conditions can affect the quality (type) and quantity (extent) of biomolecular damage such as DNA lesions. Here, we comparatively study the influence of indirect effects of CONV and UHDR on the formation of DNA strand breaks and clustered damage including densely accumulated lesions in an aerated and an anoxic dilute aqueous solution of a plasmid DNA model under low and high hydroxyl radical (•OH) scavenging conditions. Aqueous solutions of purified supercoiled plasmid DNA (pUC19) were prepared in either air- or nitrogen-saturated conditions, with Tris buffer added as the radiation-produced •OH scavenger at low and high scavenging capacities. These DNA samples were irradiated using kV X-ray systems at CONV (0.1 Gy/s) and high dose rate (HDR, 25 Gy/s) as well as UHDR (55 and 125 Gy/s) under different scavenging and environmental conditions. DNA lesions including strand breaks and clustered damage including densely accumulated lesions were quantified by gel electrophoresis and the yields of these lesions were calculated from the dose-response curve. Non-DSB clustered damage including densely accumulated lesions were evaluated by treating DNAs using bacterial endonuclease enzymes (Fpg and Nth) prior to gel electrophoresis. UHDR of 55 and 125 Gy/s induced lower amounts of both isolated strand breaks and clustered DNA damage including densely accumulated lesions at doses >40 Gy in the presence of oxygen, compared to the abundance of these lesions induced by 0.1 and 25 Gy/s irradiation under the same dose conditions. Overall, the strand break and clustered damage including densely accumulated lesions yields decreased by factors of 1.3-3.5 after UHDR. We did not observe these differences either via •OH scavenging or by removing oxygen from the solution. In addition, our results point out that the inter-track recombination reactions did not contribute to the observed dose-rate effects on DNA damage. The effects of dose rate on DNA damage are highly dependent on the total dose, as expected, but also on the •OH scavenging capacity that is employed in the aqueous DNA solutions. These important variables may be relevant in biological systems as well. On a practical level, our in vitro plasmid DNA model, which permits to precisely vary the scavenging capacity and gassing conditions (air saturated vs. N2 saturated) can help to differentiate dose-rate effects on biomolecular damage. Our results indicate that the radical-radical reactions are important in understanding the dose-rate effect on DNA damage.

Signaling Molecules in Pseudomonas aeruginosa Response to Antibiotics at Sub-Inhibitory Concentrations

Signaling Molecules are N-acylhomoserine lactones (AHLs) that form the Pseudomonas aeruginosa cell information system which determines gene expressions in a population dependent manner called quorum sensing (QS). Signal molecules, which are chemically varied, control genes expressions to antibiotics resistance, pathogenicity, motility, biofilm development, bioluminescence, secondary metabolite production, and plasmid transfer. This research was aimed to identify the signaling molecules in Pseudomonas aeruginosa response to antibiotics at sub-inhibitory concentrations. One hundred and fifty (150) clinical swab specimens were collected from urinary catheter wound and ear infection of patients and the swabs inoculated using standard microbiology method. The isolates were characterized based on the bacteriological methods such as morphology and biochemical tests. The isolates were further confirmed by species specific PCR by amplification of 16S rRNA and the amplicons were analyzed by gel electrophoresis; and further genomic sequencing was done and blast with NCBI database mining. Disc diffusion method was applied for the antibiotics susceptibility pattern. The isolates cell suspensions were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). The result of the isolation showed 22(59.46%) from wound infections, 12(32.43%) from ear infections and 3(8.11%) from urinary catheter. The isolates were Gram negative, produced β-hemolysis on blood agar and the morphology is small pigmented circular in form. The isolates showed positive result to catalase, oxidase, citrate, nitrate and indole tests. The amplification of 16S rRNA gene region resulted in the band size of 1500bp PCR product and the BLAST analysis gave 99% similarity. The resistant antibiotics susceptibility showed that Pseudomonas aeruginosa is multidrug resistant bacteria. The GC-MS results obtained from urinary catheter isolates showed four (4) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone, N-Dodecanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone. Three (3) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone were obtained from wound isolates. N-Tetradecanoyl-L-Homoserine Lactone was the only signaling molecule obtained from ear isolates. Further research should be done to develop rapid tests that could be possible to detect acyl homoserine specific to Pseudomonsa aeruginosa present to human samples and deliver results in real time. Signaling molecules inhibitors (SMI) are possible potential therapeutics that could produce the subsequent class of antibacterial drugs. The fundamental role of each signal molecule in the production of biofilms and virulent factors should also be investigated, as well as whether Pseudomonas aeruginosa needs one or more signaling molecules to form quorum sensing. Identification of the signals molecules (AHLs) of P. aeruginosa and developing signaling molecules inhibitors (SMI) can have an important prognostic, diagnostic and therapeutic value in human medicine for the treatment of P. aeruginosa infections.

Diagnostic Accuracy of Beta-2 Transferrin Gel Electrophoresis for Detecting Cerebrospinal Fluid Rhinorrhea.

Unilateral thin clear rhinorrhea (UTCR) may represent a variety of pathologies including cerebrospinal fluid (CSF) rhinorrhea. Beta-2 transferrin (B2Tf) gel electrophoresis (GE) has become the preferred testing modality due to reportedly high sensitivity (87%-100%) and specificity (71%-100%). However, there have been relatively few studies assessing its diagnostic accuracy. The purpose of this single-institution study was to determine the accuracy of B2Tf GE in detecting CSF rhinorrhea. A single-center retrospective review was conducted from 2016 and 2024 for all patients who presented with UTCR and underwent B2Tf GE. Institutional review board approval was obtained. The gold standard for diagnostic confirmation of true and false positives (TP, FP) as well as false negatives (FN) was endoscopic exploration. The gold standard for true negative (TN) was response to medical therapy. A total of 105 patients underwent 149 B2Tf GE tests. 40 (38.1%) patients were diagnosed with CSF rhinorrhea. Of the 149 B2-Tf GE tests, there were 51 TPs, 72 TNs, 20 FPs, and 6 FNs yielding 89.5% sensitivity, 78.3% specificity, 71.8% positive predictive value, and 92.3% negative predictive value, respectively. Of the false results the most common causes for error were purulent sinusitis (n = 6, 23.1%), possible mucous contamination from nose-blowing during collection (n = 3, 11.5%), patient collection error (n = 3, 11.5%), and blood contamination (n = 1, 3.8%). Although these single-institutional data demonstrate test accuracy within ranges previously reported in the literature, they also demonstrate diagnostic limitations. Future studies should explore reasons for erroneous B2Tf GE results and how these may change clinical decision-making. IV Laryngoscope, 2024.

Characterization of high Fischer ratio oligopeptides produced from casein

High Fischer ratio oligopeptides mixture is a mixed peptide system which possess many beneficial effects to the human body. Casein, a milk protein, was used as feedstock to prepare high Fischer ratio oligopeptides via enzymatic hydrolysis, adsorption and purification. Gel electrophoresis, densitometric analysis, amino acid assay and molecular weight measurement were used to visualize and quantify the prepared peptides. Casein, with Fischer ratio of 2.17, was subjected to hydrolysis by alcalase® (2.4 AU/g, pH 8.5, temperature 55 °C, enzymolysis time 2 h) and flavorzyme® (3.4 AU/g, temperature 50 °C, enzyme amount 120 μL, enzymolysis time 3 h), followed via AK-220 activated carbon adsorption (carbon-hydrolysate ratio: 1:10, adsorption time 3 h, pH 8.0, temperature 55 °C) and 3 KDa membrane ultrafiltration. In conclusion, the processing parameters for alcalase® / flavorzyme® and AK-220 activated carbon helped to obtain oligopeptides with Fischer ratio 9.87 which is significantly higher than feedstock casein F value (p < 0.05) and could potentially be used as food supplement to enhance product nutritional property.

Molecular Detection of Beta-Lactamase Genes (KPC and CTX-M) of Pseudomonas aeruginosa in Diabetic Foot Ulcers Patients

Background: diabetic foot ulcers (DFUs ) are an inflammatory disease caused by a bacterial infection such as Pseudomonas aeruginosa (P. aeruginosa), provided with genetic virulence factors. This study aims to detect KPC and CTX-M genes of P. aeruginosa isolated from chronic DFU patients. Methods: One hundred twenty DFU patients were recruited from Al-Sader Medical City and Al-Hakim General Hospital in Najaf province from September to January 2021 to collect ear swaps. The Primary identification depends on Gram stain and biochemical tests. We also used the Vitek 2 system to make another identification and examine resistance to antibiotics. A polymerase chain reaction (PCR) was used to amplify the required genes. Furthermore, gel electrophoresis was performed to show the amplified genes. Results: The results have revealed that 100 samples (83.3%) give a positive outcome, while 20 samples (16.6%)are negative for culture bacteria. Only 17(17 %) of the 100 clinical specimens showed positive tests for P. aeruginosa. However, the gene, CTX-M gene is found in 58.8% of P. aeruginosa isolates, while the KPC gene is not found in the isolates. Conclusion: Our findings suggest that P. aeruginosa is one of the most common causes of DFU infection in Iraqi patients and The levels of MDR of P. aeruginosa isolates in Najaf hospitals were undoubtedly high. Most bacterial isolates show the presence of the CTX-M gene. The presence of these genes is one of the virulence factors and probably partly explains the resistance to antibiotics.

Cryopreservation of bovine sperm causes single-strand DNA breaks that are localized in the toroidal regions of chromatin

BackgroundSperm cryopreservation is widely used in the cattle industry, as it allows for disassociating the localization of sires and the collection of semen from the timing of artificial insemination. While freeze-thawing is known to impair sperm DNA integrity, whether the damage induced consists of single- (SSB) or double-strand breaks (DSB) has not been determined. In addition, no previous study has addressed if DNA breaks preferentially reside in specific genome regions such as those forming the toroid linker regions, or are rather spread throughout the regions linked to protamines. The main aim of the present work, therefore, was to elucidate the type and localization of the DNA damage generated by cryopreservation and to evaluate its impact on artificial insemination outcomes in cattle.ResultsThe incidence of SSB and DSB was evaluated in 12 ejaculates before and after cryopreservation with the Comet assay, and the localization of the DNA breaks was assessed using pulsed-field gel electrophoresis (PFGE). Before cryopreservation, the incidence of SSB was 10.99% ± 4.62% and involved 20.56% ± 3.04% of sperm cells, whereas these figures significantly (P < 0.0001) increased up to 34.11% ± 3.48% and 53.36% ± 11.00% in frozen-thawed sperm. In contrast, no significant differences in the incidence of DSB were observed (P > 0.990) before and after cryopreservation (before: incidence of 13.91% ± 1.75% of sperm DNA affecting 56.04% ± 12.49% of sperm cells; after: incidence of 13.55% ± 1.55% of sperm DNA involving 53.36% ± 11.00% of sperm cells). Moreover, PFGE revealed that the percentage of sperm DNA fragments whose length was shorter than a toroid (< 31.5 kb) was greater (P < 0.0001) after (27.00% ± 4.26%) than before freeze-thawing (15.57% ± 4.53%). These differences indicated that the DNA breaks induced by cryopreservation affect the regions condensed in protamines, which are structured in toroids. On the other hand, in vivo fertility rates were associated to the incidence of SSB and DSB in frozen-thawed sperm (P = 0.032 and P = 0.005), but not with the size of the DNA fragments resulting from these breaks (P > 0.05).ConclusionCryopreservation of bovine sperm generates single-strand DNA breaks, which are mainly located in protamine-condensed toroidal regions. The incidence of DNA breaks in cryopreserved sperm has an impact on cattle fertility, regardless of the size of generated fragments.

Extraction and Nutritional Value of Soybean Meal Protein Isolate

The increasing protein demand driven by global population growth necessitates the search for an alternative protein source. Soybean meal (SM), with approximately 47–49% proteins, is a viable option. Soymeal protein isolate (SMPI) is a cost-efficient protein source with a well-balanced amino acid profile, making it suitable for addressing this demand. This study attempts to address the lack of information regarding the extractability and nutritional potential of SMPI obtained utilizing a weak base and recoverable solvent. In this work, the structural and compositional studies of SMPI, as well as the aqueous extractability of ammonium hydroxide (NH4OH), are investigated. Furthermore, we examined the effects of heat treatment during oil extraction, where a comparison between protein isolates from soymeal and whole soybeans was carried out. The maximum extraction yield of 24.1% was achieved by increasing the concentration of NH4OH from 0.25–1%. In comparison to the soy protein isolate from whole soybeans (SPI), the compositional analysis report revealed that SMPI had higher levels of crude protein, moisture, and ash content, but lower levels of fat and carbohydrates. Furthermore, the examination of the amino acid composition confirms the existence of vital amino acids in SMPI. The amino acid score indicates that methionine, lysine, and threonine are the limiting amino acids. SMPI and SPI share structural and functional group similarities, as demonstrated by Fourier-transform infrared spectroscopy. Gel electrophoresis using sodium dodecyl sulfate–polyacrylamide shows that the protein molecular weight distributions of SPI and SMPI are similar. This in-depth evaluation emphasizes the advantages of SM by advocating its application in other sectors beyond conventional animal feed, such as nutritional supplements and bio-based products, and by improving the environmental sustainability and global food chains.

Isolation and purification of DNA double-strand break repair intermediates for understanding complex molecular mechanisms.

Branched DNA molecules are key intermediates in the molecular pathways of DNA replication, repair and recombination. Understanding their structural details, therefore, helps to envisage the mechanisms underlying these processes. While the configurations of DNA molecules can be effectively analysed in bulk using gel electrophoresis techniques, direct visualization provides a complementary single-molecule approach to investigating branched DNA structures. However, for microscopic examination, the sample needs to be free from impurities that could obscure the molecules of interest, and free from the bulk of unwanted non-specific DNA molecules that would otherwise dominate the field of view. Additionally, in the case of recombination intermediates, the length of the DNA molecules becomes an important factor to consider since the structures can be spread over a large distance on the chromosome in vivo. As a result, apart from sample purity, efficient isolation of large-sized DNA fragments without damaging their branched structures is crucial for further analysis. These factors are illustrated by the example of DNA double-strand break repair in the bacterium E. coli. In E. coli recombination intermediates may be spread over a distance of 40 kb which constitutes less than 1% of the 4.6 Mb genome. This study reveals ways to overcome some of the technical challenges that are associated with the isolation and purification of large and complex branched DNA structures using E. coli DNA double-strand break repair intermediates. High-molecular weight and branched DNA molecules do not run into agarose gels subjected to electrophoresis. However, they can be extracted from the wells of the gels if they are agarose embedded, by using β-agarase digestion, filtration, and concentration. Furthermore, a second round of gel electrophoresis followed by purification is recommended to enhance the purity of the specific DNA samples. These preliminary findings may prove to be pioneering for various single-molecule analyses of large and complex DNA molecules of DNA replication, repair and recombination.