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Ribosome-inactivating proteins (RIPs) are highly active N-glycosidases that depurinate both bacterial and eukaryotic rRNAs, halting protein synthesis during translation. Found in a diverse spectrum of plant species and tissues, RIPs possess antifungal, antibacterial, antiviral, and insecticidal properties linked to plant defense. In this study, we investigated the physiochemical properties of RIP peptides from the Cucurbitaceae family through bioinformatics approaches. Molecular weight, isoelectric point, aliphatic index, extinction coefficient, and secondary structures were analyzed, revealing their hydrophobic nature. The novelty of this work lies in the comprehensive examination of RIPs from the Cucurbitaceae family and their potential therapeutic applications. The study also elucidated the binding interactions of Cucurbitaceae RIPs with key biological targets, including Interleukin-6 (IL-6). Strong hydrogen bond interactions between RIPs and these targets suggest potential for innovative insilico drug design and therapeutic applications, particularly in cancer treatment. Comprehensive analysis of bond lengths using Ligpolt + software provides insights for optimizing molecular interactions, offering a valuable tool for drug design and structural biology studies.

Genetic code expansion involves introducing non-canonical amino acids (ncAAs) with unique functional groups into proteins to broaden their applications. Orthogonal aminoacyl tRNA synthetase (aaRS), essential for genetic code expansion, facilitates the charging of ncAAs to tRNA. In this study, we developed a new aaRS mutant from Methanosaeta concilii tyrosyl-tRNA synthetase (Mc TyrRS) to incorporate para-azido-L-phenylalanine (AzF). The development involved initial site-specific mutations in Mc TyrRS, followed by random mutagenesis. The new aaRS mutant with amber suppression was isolated through fluorescence-activated cell sorting. The M. concilii aaRS mutant structure was further analyzed to interpret the effect of mutations. This research provides a novel orthogonal aaRS evolution pipeline for highly efficient ncAA incorporation that will contribute to developing novel aaRS from various organisms.

This experiment aimed to investigate the impact of malic acid (MA) and citric acid (CA) on the nutritional composition, fermentation quality, rumen degradation rate, and microbial diversity of a mixture of apple pomace and corn protein powder during ensiling. The experiment used apple pomace and corn protein powder as raw materials, with four groups: control group (CON), malic acid treatment group (MA, 10 g/kg), citric acid treatment group (CA, 10 g/kg), and citric acid + malic acid treatment group (MA, 10 g/kg + CA, 10 g/kg). Each group has 3 replicates, with 2 repetitions in parallel, subjected to mixed ensiling for 60 days. The results indicated: (1) Compared to the CON group, the crude protein content significantly increased in the MA, CA, and MA + CA groups (p < 0.05), with the highest content observed in the MA + CA group. The addition of MA and CA effectively reduced the water-soluble carbohydrate (WSC) content (p < 0.05). Simultaneously, the CA group showed a decreasing trend in NDFom and hemicellulose content (p = 0.08; p = 0.09). (2) Compared to the CON group, the pH significantly decreased in the MA, CA, and MA + CA groups (p < 0.01), and the three treatment groups exhibited a significant increase in lactic acid and acetic acid content (p < 0.01). The quantity of lactic acid bacteria increased significantly (p < 0.01), with the MA + CA group showing a more significant increase than the MA and CA groups (p < 0.05). (3) Compared to the CON group, the in situ dry matter disappearance (ISDMD) significantly increased in the MA, CA, and MA + CA groups (p < 0.05). All three treatment groups showed highly significant differences in in situ crude protein disappearance (ISCPD) compared to the CON group (p < 0.01). (4) Good's Coverage for all experimental groups was greater than 0.99, meeting the conditions for subsequent sequencing. Compared to the CON group, the Shannon index significantly increased in the CA group (p < 0.01), and the Simpson index increased significantly in the MA group (p < 0.05). However, there was no significant difference in the Chao index among the three treatment groups and the CON group (p > 0.05). At the genus level, the abundance of Lentilactobacillus in the MA, CA, and MA + CA groups was significantly higher than in the control group (p < 0.05). PICRUSt prediction results indicated that the metabolic functional microbial groups in the CA and MA treatment groups were significantly higher than in the CON group (p < 0.05), suggesting that the addition of MA or CA could reduce the loss of nutritional components such as protein and carbohydrates in mixed ensilage. In conclusion, the addition of malic acid and citric acid to a mixture of apple pomace and corn protein powder during ensiling reduces nutritional losses, improves fermentation quality and rumen degradation rate, enhances the diversity of the microbial community in ensiled feed, and improves microbial structure. The combined addition of malic acid and citr

The growth of material science and technology places a high importance on the creation of better processes for the synthesis of copper nanoparticles. So that, an easy, ecological, and benign process for producing copper nanoparticles (CuNPs) has been developed using candy leaf (Stevia rebaudiana) leaves aqueous extract for the first time. UV-visible spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), high-resolution transmission electron microscope (HR-TEM), Fourier transmission infrared (FTIR), and zeta potential were applied to demonstrate strong characterization for the biosynthesized stevia-CuNPs. The UV-visible absorbance at 575 nm of surface plasmon resonance (SPR) was 1.2. The particle size mean diameter was recorded as 362.3 nm with - 10.8 mV zeta potential. The HR-TEM scanning revealed 51.46-53.17 nm and spherical-shaped stevia-CuNPs surrounded by coat-shell proteins. The cytotoxicity and cytocompatibility activity assay revealed that stevia-CuNPs was safe in lower concentrations and had a significant cell viability reduction in higher concentrations. The produced stevia-CuNPs were applied as antimicrobial agents against eight pathogenic bacteria and five fungi strains. The inhibitory action of the stevia-CuNPs was more pronounced in bacteria than in fungi, and they likewise demonstrated further inhibition zones in Staphylococcus aureus (50.0 mm) than in Aspergillus flavus (55.0 mm). With inhibition zone sizes of 50.0 mm and 47.0 mm and 50 µg/ml minimum inhibitory concentration, S. aureus and A. flavus were the most inhibited pathogens. The minimum lethal effect (MLC) estimate for S. aureus was 50 µg/ml, whereas 75 µg/ml for A. flavus. The stevia-CuNPs mode of action was characterized as bactericidal/fungicidal as the ratio of MIC to MLC was estimated to be equal to or less than 2. After all, stevia-CuNPs could be used as an alternative to commercial antibiotics to solve the problem of multidrug-resistant (MDR) microorganisms.

Respiratory coronaviruses (RCoVs) significantly threaten human health, necessitating the development of an ex vivo respiratory culture system for investigating RCoVs infection. Here, we successfully generated a porcine precision-cut lung slices (PCLSs) culture system, containing all resident lung cell types in their natural arrangement. Next, this culture system was inoculated with a porcine respiratory coronavirus (PRCV), exhibiting clinical features akin to humans who were infected by SARS-CoV-2. The results demonstrated that PRCV efficiently infected and replicated within PCLSs, targeting ciliated cells in the bronchioles, terminal bronchioles, respiratory bronchioles, and pulmonary alveoli. Additionally, through RNA-Seq analysis of the innate immune response in PCLSs following PRCV infection, expression levels of interferons, inflammatory cytokines and IFN stimulated genes were significantly upregulated. This ex vivo model may not only offer new insights into PRCV infection in the porcine respiratory tract but also serve as a valuable tool for studying human respiratory CoVs infection.

African swine fever virus (ASFV) is a highly pathogenic and rapidly disseminated virus with strong viability in the environment, suggesting the importance of environmental detection for prevention and control in all the pig industry. However, the detection results of environmental swabs cannot always reflect the accurate status of viral pollution, leading to persistent ASFV environmental contamination. In this study, we developed an ASFV eluant with higher environmental ASFV detection efficiency relative to 0.85% saline solution, which obtains the patent certificate issued by the China Intellectual Property Office (patent number:202010976050.9). qPCR analysis showed that in the environmental swab samples, the number of viral copies was 100 times higher for the ASFV eluant treatment than the traditional eluant treatment (0.85% saline solution). And besides, the high sensitivity of the ASFV eluant had be verified in a slaughterhouse environmental sampling detection. In soil samples, the ASFV eluent showed the same extraction effect as the TIANamp Soil DNA Kit, in contrast to no extraction effect for 0.85% saline solution. Simultaneously, this eluent could protect ASFV from degradation and allow the transportation of samples at ambient temperature without refrigeration. In clinical practice, we monitored the environmental contamination condition of the ASFV in a large-scale pig farm. The results shown that the ASFV load decreased after every disinfection in environment. This study provides an effective solution for surveilling the potential threat of ASFV in environment.

In the present study, low- and high-molecular-weight hyaluronic acids (LMW-HA and HMW-HA) were synthesized in vitro by truncated Streptococcus equisimilis hyaluronan synthases (SeHAS). The enzyme kinetic parameters were determined for each enzyme variant. The MW, structure, dispersity, and biological activity of polymers were determined by electrophoresis, FTIR spectroscopy, carbazole, cell proliferation, and cell migration assay, respectively. The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 µg_HA µg_enzyme^-1 min^-1 for SeHAS, HAS₁₂₃, HAS₂₃, and HAS_Intra, respectively. The results revealed SeHAS produced a polydisperse HMW-HA (268 kDa), while HAS₁₂₃ and HAS₂₃ produced a polydisperse LMW-HA (< 30 kDa). Interestingly, HAS_Intra produced a low-disperse LMW-HA. Kinetics studies revealed the truncated variants displayed increased K_m values for two substrates when compared to the wild-type enzyme. Biological assessments indicated all LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest cell migration effect at 10 µg/mL, while at 200 µg/mL, both LMW- and HMW-HAs postponed the healing recovery rate. The study elucidated that the transmembrane domains (TMDs) of SeHAS affect the enzyme kinetics, HA-titer, HA-size, and HA-dispersity. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.

Phoenix dactylifera L. and its wastes are known to be high in nutrients that are beneficial to human health. The study aimed to evaluate the antimicrobial, antibiofilm, and antiviral properties of Phoenix dactylifera L. pits extract (PDPE) in vitro. Gas chromatography-mass spectrometry (GC-MS) analysis indicated phenol, 2,5-bis(1,1-dimethyl ethyl), tetradecanoic acid, octaethylene glycol monododecyl ether, á-D-glucopyranosiduronic acid, and heptaethylene glycol monododecyl ether existence. The PDPE influenced pathogenic microorganisms, with inhibition zone diameters (IZDs) ranging from 10.0 to 35.0 mm. Staphylococcus aureus ATCC 5638 had the highest IZD, while Salmonella typhi DSM 17058 and Shigella sonnei DSM 5570 had the lowest. The antifungal effect observed only in spore failure or conidia formation. PDPE showed a 100% antibacterial spectrum against bacteria, with MIC values between 250 and 1000 µg/ml. MIC was only indicated with S. aureus of 500 µg/ml. MBC values ranged from 500 to 1000 g/ml, with MBC values of 500 g/ml for B. cereus, E. faecalis, S. typhi, and S. sonnei. The activity was 66.7% at 500 µg/ml, further concentrations of 125-250 g/ml had no antibacterial effect. PDPE biofilm inhibition % had the highest percentage of inhibition (98.59%) with S. aureus, B. cereus (94.12%), and E. coli (74.46%). With 50% (CC₅₀) viral activity, the highest non-toxic PDPE dose was found to be at 123.0 µg/ml.

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10-15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1-2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Klebsiella pneumoniae (K. pneumoniae) is a member of the ESKAPE group and is responsible for severe community and healthcare-associated infections. Certain Klebsiella species have very similar phenotypes, which presents a challenge in identifying K. pneumoniae. Multidrug-resistant K. pneumoniae is also a serious global problem that needs to be addressed. A total of 190 isolates were isolated from urine (n = 69), respiratory (n = 52), wound (n = 48) and blood (n = 21) samples collected from various hospitals in the Al-Qassim, Saudi Arabia, between March 2021 and October 2022. Our study aimed to rapidly and accurately detect K. pneumoniae using the Peptide Mass Fingerprinting (PMF) technique, confirmed by real-time PCR. Additionally, screening for antibiotic susceptibility and resistance was conducted. The primary methods for identifying K. pneumoniae isolates were culture, Gram staining, and the Vitek® 2 ID Compact system. An automated MALDI Biotyper (MBT) instrument was used for proteome identification, which was subsequently confirmed using SYBR green real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Vitek® 2 AST-GN66 cards were utilized to evaluate the antimicrobial sensitivity of K. pneumoniae isolates. According to our results, Vitek® 2 Compact accurately identified 178 out of 190 (93.68%) K. pneumoniae isolates, while the PMF technique correctly detected 188 out of 190 (98.95%) isolates with a score value of 2.00 or higher. Principal component analysis was conducted using MBT Compass software to classify K. pneumoniae isolates based on their structure. Based on the analysis of the single peak intensities generated by MBT, the highest peak values were found at 3444, 5022, 5525, 6847, and 7537 m/z. K. pneumoniae gene testing confirmed the PMF results, with 90.53% detecting entrobactin, 70% detecting 16 S rRNA, and 32.63% detecting ferric iron uptake. The resistance of the K. pneumoniae isolates to antibiotics was as follows: 64.75% for cefazolin, 62.63% for trimethoprim/sulfamethoxazole, 59.45% for ampicillin, 58.42% for cefoxitin, 57.37% for ceftriaxone, 53.68% for cefepime, 52.11% for ampicillin-sulbactam, 50.53% for ceftazidime, 52.11% for ertapenem, and 49.47% for imipenem. Based on the results of the double-disk synergy test, 93 out of 190 (48.95%) K. pneumoniae isolates were extended-spectrum beta-lactamase. In conclusion, PMF is a powerful analytical technique used to identify K. pneumoniae isolates from clinical samples based on their proteomic characteristics. K. pneumoniae isolates have shown increasing resistance to antibiotics from different classes, including carbapenem, which poses a significant threat to human health as these infections may become difficult to treat.