A chemiluminescent immunoassay for human serum Cystatin C (Cys C) was established using a direct-antibody sandwich model. The immunoassay kit uses magnetic separation technology, using magnetic particles as the reaction solid phase, alkaline phosphatase as the marker enzyme, and a new chemiluminescent substrate APLS as the substrate. It has the characteristics of high sensitivity and short reaction time. This product uses high-affinity antibodies, resulting in a high specificity. The established method showed good accuracy, uniformity, and stability. The limit of detection was 2.39 ng/mL. The intra-assay coefficient of variation (CV) was 3.36%–6.00%, the interassay CV was 4.12%–5.35%, and the recovery rate was 99.07%. The correlation coefficient (r) of Cys-C kit was 0.999388 ≥ 0.9900. The accuracy of the developed method was tested by automatic chemiluminescence instrument (P > 0.05). The lowest titer was 0.92500, and the highest was 1.10000. The developed method showed a good correlation with the product from Roche by comparing these two kits in 240 clinical samples from China. In total, 1392 clinical patient from China samples were measured using the reagent kit developed in this study.

Target identification is an essential part of the drug discovery and development process, and its efficacy plays a crucial role in the success of any given therapy. Although protein target identification research can be challenging, two main approaches can help researchers make significant discoveries: affinity-based pull-down and label-free methods. Affinity-based pull-down methods use small molecules conjugated with tags to selectively isolate target proteins, while label-free methods utilize small molecules in their natural state to identify targets. Target identification strategy selection is essential to the success of any drug discovery process and must be carefully considered when determining how to best pursue a specific project. This paper provides an overview of the current target identification approaches in drug discovery related to experimental biological assays, focusing primarily on affinity-based pull-down and label-free approaches, and discusses their main limitations and advantages.

BackgroundThe major safety concern of the clinical application of wild type FGF19 (FGF19WT) emerges given that its extended treatment causes hepatocellular carcinoma. Therefore, we previously generated a safer FGF19 variant - FGF19ΔKLB, which have same effects on glycemic control and bile acid production but much less mitogenic activity. However, it remains unclear as to whether FGF19ΔKLB ameliorates intrahepatic cholestasis.ResultsWe found that, similar to that of FGF19WT, the chronic administration of FGF19ΔKLB protects mice from cholestatic liver injury in these two models. The therapeutic benefits of FGF19ΔKLB on cholestatic liver damage are attributable, according to the following mechanistic investigation, to the reduction of BA production, liver inflammation, and fibrosis. More importantly, FGF19ΔKLB did not induce any tumorigenesis effects during its prolonged treatment.ConclusionsTogether, our findings raise hope that FGF19ΔKLB may represent a useful therapeutic strategy for the treatment of intrahepatic cholestasis.

Metal nanoparticles exhibit excellent antifungal abilities and are seen as a good substitute for controlling different kinds of fungi. Of all known taxa, cyanobacteria have received significant consideration as nanobiofactories, as a result of the cellular assimilation of heavy metals from the environment. The cellular bioactive enzymes, polysaccharides and pigments can be used as reducers and coatings during biosynthesis. The probability of the antifungal activity of selenium nanoparticles (SeNPs) to prevent plant fungi that can affect humans was evaluated and a toxic Iranian cyanobacterial strain of Desmonostoc alborizicum was used to study the biotechnology of SeNP synthesis for the first time. Characterization of nanoparticles with a UV-Vis spectrophotometer showed the formation of SeNPs in the range of 271–275 nm with the appearance of an orange color. Morphological examination of nanoparticles with Transmission Electron Microscopy (TEM), revealed the spherical shape of nanoparticles. The results of X-Ray Diffraction (XRD) showed 7 peaks and a hexagonal structure of average crystal size equal to 58.8 nm. The dispersion index of SeNPs was reported as 0.635, which indicated the homogeneity of the nanoparticle droplet size. The zeta potential of the nanoparticles was + 22.7. Fourier-transform infrared spectroscopy (FTIR) analysis exhibited a sharp and intense peak located at the wave number of 404 cm− 1, related to the SeNPs synthesized in this research. The results of the antifungal activity of SeNPs showed among the investigated fungi, Pythium ultimum had the highest resistance to SeNPs (14.66 ± 0.52 µg/ml), while Alternaria alternata showed the highest sensitivity (9.66 ± 0.51 µg/ml) (p < 0.05). To the best of our knowledge this is the first report concerning the characterization and antifungal screening of SeNPs biosynthesized by Iranian cyanobacteria, which could be used as effective candidates in medical applications.

BackgroundHelicobacter pylori cause a variety of gastric malignancies, gastric ulcers, and cause erosive diseases. The extreme nature of the bacterium and the implantation of this bacterium protects it against designing a potent drug against it. Therefore, employing a precise and effective design for a more safe and stable antigenic vaccine against this pathogen can effectively control its associated infections. This study, aimed at improving the design of multiple subunit vaccines against H. pylori, adopts multiple immunoinformatics approaches in combination with other computational approaches.ResultsIn this regard, 10 HTL, and 11 CTL epitopes were employed based on appropriate adopted MHC binding scores and c-terminal cut-off scores of 4 main selected proteins (APO, LeoA, IceA1, and IceA2). An adjuvant was added to the N end of the vaccine to achieve higher stability. For validation, immunogenicity and sensitization of physicochemical analyses were performed. The vaccine could be antigenic with significantly strong interactions with TOLK-2, 4, 5, and 9 receptors. The designed vaccine was subjected to Gromacs simulation and immune response prediction modelling that confirmed expression and immune-stimulating response efficiency. Besides, the designed vaccine showed better interactions with TLK-9.ConclusionsBased on our analyses, although the suggested vaccine could induce a clear response against H. pylori, precise laboratory validation is required to confirm its immunogenicity and safety status.

BackgroundCutaneotrichosporon oleaginosus is an oleaginous yeast that can produce up to 80% lipid per dry weight. Its high capacity for the biosynthesis of single cell oil makes it highly interesting for the production of engineered lipids or oleochemicals for industrial applications. However, the genetic toolbox for metabolic engineering of this non-conventional yeast has not yet been systematically expanded. Only three long endogenous promoter sequences have been used for heterologous gene expression, further three dominant and one auxotrophic marker have been established.ResultsIn this study, the structure of putative endogenous promoter sequences was analyzed based on more than 280 highly expressed genes. The identified motifs of regulatory elements and translational initiation sites were used to annotate the four endogenous putative promoter sequences D9FADp, UBIp, PPIp, and 60Sp. The promoter sequences were tested in a construct regulating the known dominant marker hygromycin B phosphotransferase. The four newly described promoters and the previously established GAPDHp successfully initiated expression of the resistance gene and PPIp was selected for further marker development. The geneticin G418 resistance (aminoglycoside 3’-phosphotransferase, APH) and the nourseothricin resistance gene N-acetyl transferase (NAT) were tested for applicability in C. oleaginosus. Both markers showed high transformation efficiency, positive rate, and were compatible for combined use in a successive and simultaneous manner.ConclusionsThe implementation of four endogenous promoters and one novel dominant resistance markers for C. oleaginosus opens up new opportunities for genetic engineering and strain development. In combination with recently developed methods for targeted genomic integration, the established toolbox allows a wide spectrum of new strategies for genetic and metabolic engineering of the industrially highly relevant yeast.

BackgroundCartilage defects are common sports injuries without significant treatment. Articular cartilage with inferior regenerative potential resulted in the poor formation of hyaline cartilage in defects. Acellular matrix scaffolds provide a microenvironment and biochemical properties similar to those of native tissues and are widely used for tissue regeneration. Therefore, we aimed to design a novel acellular cartilage matrix scaffold (ACS) for cartilage regeneration and hyaline-like cartilage formation.MethodsFour types of cartilage injury models, including full-thickness cartilage defects (6.5 and 8.5 mm in diameter and 2.5 mm in depth) and osteochondral defects (6.5 and 8.5 mm in diameter and 5 mm in depth), were constructed in the trochlear groove of the right femurs of pigs (n = 32, female, 25–40 kg). The pigs were divided into 8 groups (4 in each group) based on post-surgery treatment differences. was assessed by macroscopic appearance, magnetic resonance imaging (MRI), micro–computed tomography (micro-CT), and histologic and immunohistochemistry tests.ResultsAt 6 months, the ACS-implanted group exhibited better defect filling and a greater number of chondrocyte-like cells in the defect area than the blank groups. MRI and micro-CT imaging evaluations revealed that ACS implantation was an effective treatment for cartilage regeneration. The immunohistochemistry results suggested that more hyaline-like cartilage was generated in the defects of the ACS-implanted group.ConclusionsACS implantation promoted cartilage repair in full-thickness cartilage defects and osteochondral defects with increased hyaline-like cartilage formation at the 6-month follow-up.

BackgroundBiofuel research that aims to optimize growth conditions in microalgae is critically important. Chlamydomonas reinhardtii is a green microalga that offers advantages for biofuel production research. This study compares the effects of nitrogen-, sulfur-, and nitrogen and sulfur- deprivations on the C. reinhardtii starchless mutant cc5373-sta6. Specifically, it compares growth, lipid body accumulation, and expression levels of acetyl-CoA carboxylase (ACC) and phosphoenolpyruvate carboxylase (PEPC).ResultsAmong nutrient-deprived cells, TAP-S cells showed significantly higher total chlorophyll, cell density, and protein content at day 6 (p < 0.05). Confocal analysis showed a significantly higher number of lipid bodies in cells subjected to nutrient deprivation than in the control over the course of six days; N deprivation for six days significantly increased the size of lipid bodies (p < 0.01). In comparison with the control, significantly higher ACC expression was observed after 8 and 24 h of NS deprivation and only after 24 h with N deprivation. On the other hand, ACC and PEPC expression at 8 and 24 h of S deprivation was not significantly different from that in the control. A significantly lower PEPC expression was observed after 8 h of N and NS deprivation (p < 0.01), but a significantly higher PEPC expression was observed after 24 h (p < 0.01).ConclusionsBased on our findings, it would be optimum to cultivate cc5373-sta6 cells in nutrient deprived conditions (-N, -S or –NS) for four days; whereby there is cell growth, and both a high number of lipid bodies and a larger size of lipid bodies produced.

BackgroundLamotrigine is an effective antiseizure medication that can be used in the management of focal and generalized epilepsies in pediatric patients. This study was conducted to quantify and compare the solubility of lamotrigine in age-specific biorelevant media that simulated the fasted and fed conditions of the gastric and intestinal environments in pediatrics and adults. Another aim was to predict how traditional, re-formulated, modified, and new oral formulations would behave in the gastric and intestinal environments across different age groups.MethodsSolubility studies of lamotrigine were conducted in 16 different age-specific biorelevant media over the pH range and temperature specified by the current biopharmaceutical classification system-based criteria. The age-specific biorelevant media simulated the environments in the stomach and proximal gastrointestinal tract in both fasted and fed conditions of adults and pediatric sub-populations. The solubility of lamotrigine was determined using a pre-validated HPLC-UV method.ResultsLamotrigine showed low solubility in the 16 age-specific biorelevant media as indicated by a dose number of > 1. There were significant age-specific variabilities in the solubility of lamotrigine in the different age-specific biorelevant media. Pediatric/adult solubility ratios of lamotrigine fell outside the 80-125% range in 6 (50.0%) and were borderline in 3 (25.0%) out of the 12 compared media. These ratios indicated that the solubility of lamotrigine showed considerable differences in 9 out of the 12 (75.0%) of the compared media.ConclusionFuture studies are still needed to generate more pediatric biopharmaceutical data to help understand the performances of oral dosage forms in pediatric sub-populations.

BackgroundClassical swine fever (CSF) is a fatal contagious disease affecting pigs caused by classical swine fever virus (CSFV). The disease can be transmitted by pigs and wild boars, and it is difficult to prevent and control. To obtain necessary information to establish the CSFV resistant animals in a future study, we designed lentiviral vector-delivered short hairpin RNAs (shRNAs) targeting the conserved domain III of the internal ribosomal entry site (IRES) of the CSFV genomic RNA.ResultsFirst, we confirmed the effects of siRNAs on CSFV-IRES activity. We observed significant inhibition of CSFV-IRES activity by si42 (domain IIIa), si107 (domain IIIc), and si198 (domain IIIf) in SK-L cells and si56 (domain IIIb), si142 (domain IIId1) and si198 in HEK293 cells without affecting the amount of luciferase RNA. Next, we constructed lentiviral vectors expressing shRNA based on siRNA sequences. Treatment with shRNA-expressing lentivirus was examined at 7 and 14 days post infection in SK-L cells and HEK293 cells, and CSFV-IRES was significantly suppressed at 14 days (sh42) post infection in HEK293 cells without significant cytotoxicity. Next, we examined the silencing effect of siRNA on CSFV replicon RNA and observed a significant effect by si198 after 2 days of treatment and by shRNA-expressing lentivirus (sh56, sh142, and sh198) infection after 14 days of treatment. Treatment of sh198-expressing lentivirus significantly suppressed CSFV infection at 3 days after infection.ConclusionThe IRES targeting sh198 expressing lentivirus vector can be a candidate tool for CSFV infection control.